Heterotrophic cultivation of hydrocarbon-producing microalgae

ABSTRACT

The invention discloses novel methods of producing hydrocarbons through heterotrophic cultivation of  Botryococcus braunii . Also provided are novel hydrocarbon compositions. A preferred species for engineering is the microalgae species  Botryococcus braunii . Additional methods of cultivation include providing certain nutrient sources.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/078,246, filed Jul. 3, 2008, which is hereby incorporated byreference in its entirety.

GOVERNMENT INTERESTS

This invention was made with United States Government support underCooperative Agreement Award Number 70NANB7H7002 awarded by the NationalInstitute of Standards and Technology (NIST).

The United States Government has certain rights in the invention.

FIELD OF THE DISCLOSED INVENTION

This disclosure relates to hydrocarbon compositions and the means fortheir production. Compositions containing one or more novel hydrocarbonsproduced by one or more microorganisms, optionally with additionalmodification in vitro, are disclosed herein. Also disclosed are methodsfor the preparation of the hydrocarbon compositions, for example byheterotrophic growth of microorganisms, such as the microalgaeBotryococcus braunii. Also disclosed are methods of geneticallyengineering hydrocarbon-producing microalgae.

BACKGROUND

Fossil fuel is a general term for buried combustible geologic depositsof organic materials, formed from decayed plants and animals that havebeen converted to crude oil, coal, natural gas, or heavy oils byexposure to heat and pressure in the earth's crust over hundreds ofmillions of years.

In common dialogue, fossil fuel, also known as mineral fuel, is usedsynonymously with other hydrocarbon-containing natural resources such ascoal, oil and natural gas. The utilization of fossil fuels has enabledlarge-scale industrial development and largely supplanted water drivenmills, as well as the combustion of wood or peat for heat. Fossil fuelsare a finite, non-renewable resource.

When generating electricity, energy from the combustion of fossil fuelsis often used to power a turbine. Older generators often used steamgenerated by the burning of the fuel to turn the turbine, but in newerpower plants the gases produced by burning of the fuel turn a gasturbine directly. With global modernization in the 20th and 21stcenturies, the thirst for energy from fossil fuels, especially gasolinederived from oil, is one of the causes of major regional and globalconflicts.

The burning of fossil fuels by humans is the largest source of emissionsof carbon dioxide, which is one of the greenhouse gases that allowsradiative forcing and contributes to global warming. In the UnitedStates, more than 90% of greenhouse gas emissions come from thecombustion of fossil fuels. In addition other air pollutants, such asnitrogen oxides, sulfur dioxide, VOCs, and heavy metals are produced.

Human activity raises levels of greenhouse gases primarily by releasingcarbon dioxide from fossil fuel combustion, but other gases, e.g.methane, are not negligible. The concentrations of several greenhousegases have increased over time due to human activities, such as burningof fossil fuels and deforestation leading to higher carbon dioxideconcentrations. According to the global warming hypothesis, greenhousegases from industry and agriculture have played a major role in therecently observed global warming.

BRIEF SUMMARY OF THE DISCLOSED INVENTION

In certain embodiments, the invention provides a method for culturingBotryococcus braunii microalgae heterotrophically. The method entails:

-   -   (a) providing culture media that includes a fixed carbon source        in a fermentor;    -   (b) inoculating the fermentor with a strain of Botryococcus        braunii microalgae capable of metabolizing the fixed carbon        source;    -   (c) culturing the microalgae in heterotrophic conditions for a        period of time sufficient to produce growth and/or propagation        of the microalgae, wherein the fermentor does not allow light to        strike the microalgae.

In particular embodiments, the conditions within the fermentor are suchthat the microalgae generally do not carry out photosynthesis duringculturing.

The fixed carbon source used in the method can be a carbohydrate, suchas glucose, mannose, galactose, or fructose, but is not limited to such.An exemplary non-carbohydrate carbon source useful in the invention isglycerol. Such suitable carbon sources can be used individually or incombination.

In certain embodiments, the culturing is carried out for a period oftime sufficient to produce growth and/or propagation of the microalgaewhereby the dry cell weight of microalgae at the end of culturing (ascompared to the approximate dry cell weight of microalgae in theinoculum) is increased by at least about 2-, about 3-, about 4-, about5-, about 6-, about 7-, about 8-, about 9-, or about 10-fold or more, orby an amount within any range having any of these values as endpoints.

In particular embodiments, the culture medium can be provided with acomplex nitrogen source before or during culturing. Exemplary complexnitrogen sources that are useful in the invention include urea,hydrolysate casein, and a combination thereof.

The inoculum added to the fermentor can be produced, in particularembodiments, by culture of B. braunii in the dark for at least onepassage prior to addition to the fermentor. The inoculum can be producedby prior culture in the dark for a plurality of passages, e.g., 2passages, 3 passages, 4 passages, or 5 or more passages. In certainembodiments, after culturing the microalgae in the fermentor for aperiod of time in the dark, all or a portion of the microalgae can betransferred to a further fermentor, where the microalgae can be furthercultured for a period of time, wherein the further fermentor does notallow light to strike the microalgae.

Another aspect of the invention is a method of producing hydrocarbonsfrom Botryococcus braunii microalgae that have be culturedheterotrophically according to the above method of the invention.Hydrocarbons are produced by culturing, according to this method, for aperiod of time to generate microalgal biomass, and extractinghydrocarbons from the microalgal biomass. Any suitable extraction methodcan be employed, such as hexane extraction, pressing biomass, and invivo extraction. In particular embodiments, the method can additionallyinclude separating different species of extracted hydrocarbons, e.g., ina fractional distillation column.

The invention also provides a culture of Botryococcus braunii microalgaeproduced according to the above culture method of the invention, as wellas a hydrocarbon extract produced from a microalgal biomass that isproduced according to this culture method.

In certain embodiments of the culture method of the invention, the drycell weight of the microalgae is greater than the dry cell weight of thesame strain of microalgae cultured in the presence of light, with allother culture conditions being the same. The dry cell weight ofmicroalgae grown using a fixed carbon source in the dark can exceed thedry cell weight of microalgae grown using the same fixed carbon sourcein the light by at least: about 2-, about 3-, about 4-, about 5-fold ormore, or by an amount within any range having any of these values asendpoints.

BRIEF DESCRIPTION OF THE DRAWINGS

Not Applicable

DETAILED DESCRIPTION OF THE DISCLOSED INVENTION Definitions

“Active in microalgae” means a nucleic acid that is functional inmicroalgae. For example, a promoter that has been used to drive anantibiotic resistance gene to impart antibiotic resistance to atransgenic microalgae is active in microalgae. Examples of promotersactive in microalgae are promoters endogenous to certain algae speciesand promoters found in plant viruses.

“Aqueous fraction” refers to the portion, or fraction, of a materialthat is more soluble in an aqueous phase in comparison to a hydrophobicphase. An aqueous phase is readily water soluble.

“Axenic” means a culture of an organism that is free from contaminationby other living organisms.

“Fermentor” or “bioreactor” means an enclosure or partial enclosure inwhich cells are cultured, optionally in suspension. A fermentor orbioreactor of the disclosure includes non-limiting embodiments such asan enclosure or partial enclosure which permits cultured cells to beexposed to light or which allows the cells to be cultured withoutexposure to light. The term “fermenter” is either synonymous with“fermentor” or refers to a microbial organism that causes fermentation.The interpretation of “fermenter” is as consistent with the context inwhich the term is used.

The term “biomass” refers to material produced by growth and/orpropagation of cells. Biomass may contain cells and/or intracellularcontents as well as extracellular material. Extracellular materialincludes, but is not limited to, compounds secreted by a cell.

“Carbohydrate” refers to one or more molecules usually consisting ofcarbon, hydrogen, and oxygen. Representative carbohydrates are glucose,xylose, and glycerol. The term “carbohydrate” as used herein can referto a mixture of different carbohydrate species such as depolymerizedcellulose, which comprises glucose, xylose, and partially depolymerizedcellulose fragments such as oligosaccharides and disaccharides, as wellas lignin.

“Carbohydrate transporter” refers to a polypeptide located in oradjacent to a lipid bilayer and facilitates the transport ofcarbohydrates across the lipid bilayer.

As used herein, a “catalyst” refers to an agent, such as a molecule ormacromolecular complex, capable of facilitating or promoting a chemicalreaction of a reactant to a product without becoming a part of theproduct. A catalyst thus increases the rate of a reaction, after which,the catalyst may act on another reactant to form the product. A catalystgenerally lowers the overall activation energy required for the reactionsuch that it proceeds more quickly or at a lower temperature. Thus areaction equilibrium may be more quickly attained. Examples of catalystsinclude enzymes, which are biological catalysts, and heat, which is anon-biological catalyst.

“Cell material” refers to material containing cells and/or intra- andextracellular contents, such as from the disruption of cells.Unprocessed material contains both hydrophobic and aqueous fractionsfrom cells. Cell material includes biomass as well as disrupted orhomogenized biomass.

“Conditions favorable to cell division” means conditions in which cellsdivide at least once every 72 hours.

The term “heterotrophic conditions” refers to the presence of at leastone fixed carbon source and the absence of light during culturing.

The term “co-culture”, and variants thereof such as “co-cultivate”,refer to the presence of two or more types of cells in the samefermentor or bioreactor. The two or more types of cells may both bemicroorganisms, such as microalgae, or may be a microalgal cell culturedwith a different cell type. The culture conditions may be those thatfoster growth and/or propagation of the two or more cell types or thosethat facilitate growth and/or proliferation of one, or a subset, of thetwo or more cells while maintaining cellular growth for the remainder.

The phrase “covalently modifies”, and variants thereof, refer to theformation of, removal or, or alteration in, one or more covalent bondsin a molecule. In the practice of the disclosed invention, the formationof, removal of, or alteration in, a covalent bond of a hydrocarbonmolecule is expressly contemplated and disclosed.

The term “cultivated”, and variants thereof, refer to the intentionalfostering of growth (increases in cell size, cellular contents, and/orcellular activity) and/or propagation (increases in cell numbers viamitosis) of one or more cells by use of intended culture conditions. Thecombination of both growth and propagation may be termed proliferation.The one or more cells may be those of a microorganism, such asmicroalgae. Examples of intended conditions include the use of a definedmedium (with known characteristics such as pH, ionic strength, andcarbon source), specified temperature, oxygen tension, carbon dioxidelevels, and growth in a fermentor or bioreactor. The term does not referto the growth of microorganisms in nature or otherwise without directhuman intervention, such as natural growth of an organism thatultimately becomes fossilized to produce geological crude oil.

“Distillation column” means a device for separating hydrocarbons basedon evaporation temperature, such as within a facility for refining crudeoil into gasoline.

“Exogenous gene” refers to a nucleic acid transformed into a cell. Atransformed cell may be referred to as a recombinant cell, into whichadditional exogenous gene(s) may be introduced. The exogenous gene maybe from a different species (and so heterologous), or from the samespecies (and so homologous) relative to the cell being transformed. Inthe case of a homologous gene, it occupies a different location in thegenome of the cell relative to the endogenous copy of the gene. Theexogenous gene may be present in more than one copy in the cell. Theexogenous gene may be maintained in a cell as an insertion into thegenome or as an episomal molecule.

“Exogenously provided” describes a molecule provided to the culturemedia of a cell culture.

“Fixed carbon source” means molecule(s) containing carbon, preferablyorganic, that are present at ambient temperature and pressure in solidor liquid form.

“Homogenate” means biomass that has been disrupted.

“Hydrophobic fraction” refers to the portion, or fraction, of a materialthat is more soluble in a hydrophobic phase in comparison to an aqueousphase. A hydrophobic fraction is substantially insoluble in water andusually non-polar.

As used herein, “hydrocarbon” refers to: (a) a molecule containing onlyhydrogen and carbon atoms wherein the carbon atoms are covalently linkedto form a linear, branched, cyclic, or partially cyclic backbone towhich the hydrogen atoms are attached; or (b) a molecule that onlyprimarily contains hydrogen and carbon atoms and that can be convertedto contain only hydrogen and carbon atoms by one or two chemicalreactions. Nonlimiting examples of the latter include hydrocarbonscontaining an oxygen atom between one carbon and one hydrogen atom toform an alcohol molecule, as well as aldehydes containing a singleoxygen atom. Methods for the reduction of alcohols to hydrocarbonscontaining only carbon and hydrogen atoms are well known. Anotherexample of a hydrocarbon is an ester, in which an organic group replacesa hydrogen atom (or more than one) in an oxygen acid. The molecularstructure of hydrocarbon compounds varies from the simplest, in the formof methane (CH₄), which is a constituent of natural gas, to the veryheavy and very complex, such as some molecules such as asphaltenes foundin crude oil, petroleum, and bitumens. Hydrocarbons may be in gaseous,liquid, or solid form, or any combination of these forms, and may haveone or more double or triple bonds between adjacent carbon atoms in thebackbone. Accordingly, the term includes linear, branched, cyclic, orpartially cyclic alkanes, alkenes, lipids, and paraffin. Examplesinclude propane, butane, pentane, hexane, octane, squalene andcarotenoids.

“Hydrocarbon modification enzyme” refers to an enzyme that alters thecovalent structure of a hydrocarbon. An example of a hydrocarbonmodification enzyme is an aldehyde decarbonylase.

The term “hydrogen:carbon ratio” refers to the ratio of hydrogen atomsto carbon atoms in a molecule on an atom-to-atom basis. The ratio may beused to refer to the number of carbon and hydrogen atoms in ahydrocarbon molecule. For example, the hydrocarbon with the highestratio is methane CH₄ (4:1).

The term “in situ” means “in place” or “in its original position”. Forexample, a culture containing a first microalgae secreting a catalystand a second microorganism secreting a substrate, wherein the first andsecond cell types produce the components necessary for a particularchemical reaction to occur in situ in the co-culture without requiringfurther separation or processing of the materials.

“Microalgae” means a microbial organism that is capable of performingphotosynthesis. Microalgae include obligate photoautotrophs, whichcannot metabolize a fixed carbon source as energy, as well asheterotrophs, which can live solely off of light, solely off of a fixedcarbon source, or a combination of the two. Microalgae can refer tounicellular organisms that separate from sister cells shortly after celldivision, such as Chlamydomonas, and can also refer to microbes such as,for example, Volvox, which is a simple multicellular photosyntheticmicrobe of two distinct cell types. “Microalgae” can also refer to cellssuch as Botryococcus, which associate with each other throughextracellular matrices made of hydrocarbons and biopolymers such aspolysaccharides. “Microalgae” also includes other microbialphotosynthetic organisms that exhibit cell-cell adhesion, such asAgmenellum, Anabaena, and Pyrobotrys.

“Mutagenize” means to alter the sequence of the genome of an organism.Mutagenesis can be through random means, such as chemical mutagenesis orvector insertion. A vector used for insertion can contain only ascreenable or selectable marker, or can also contain a nucleic acidsequence designed to express a gene, such as a cDNA or an antisense orRNAi construct. Mutagenesis can also be through directed means, such asthrough homologous recombination.

“Wastewater” is watery waste which typically contains washing water,laundry waste, faeces, urine and other liquid or semi-liquid wastes. Itincludes some forms of municipal waste as well as secondarily treatedsewage.

“Naturally produced” describes a compound that can be produced by awild-type organism.

“Photobioreactor” or “photofermentor” refers to a container, at leastpart of which is at least partially transparent or partially open,thereby allowing light to pass through, in which one or more microalgaecells are cultured. Photobioreactors or photofermentors may be closed,as in the instance of a polyethylene bag or Erlenmeyer flask, or may beopen to the environment, as in the instance of an outdoor pond.

“Port”, in the context of a fermentor, bioreactor, photofermentor, orphotobioreactor, refers to an opening in the photobioreactor that allowsinflux or efflux of materials such as gases, liquids, and cells. Portsare usually connected to tubing leading from the fermentor, bioreactor,photofermentor, or photobioreactor.

The term “transport protein” refers to a polypeptide located in oradjacent to a lipid bilayer and facilitates the transport of moleculesor ions across the lipid bilayer.

“Vessel” refers to a container for use in performing biochemicalreactions, chemical separations, microbial cultivation, and otherfunctions.

For sequence comparison to determine percent nucleotide or amino acididentity, typically one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (at the web addresswww.ncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra.). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. For identifying whether a nucleicacid or polypeptide is within the scope of the invention, the defaultparameters of the BLAST programs are suitable. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a word length(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. TheTBLATN program (using protein sequence for nucleotide sequence) uses asdefaults a word length (W) of 3, an expectation (E) of 10, and aBLOSUM62 scoring matrix. (see Henikoff & Henikoff, Proc. Natl. Acad.Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

I General

The invention is premised in part on the insight that certainmicroorganisms can be used to produce hydrocarbon compositionseconomically and in large quantities for use in the transportation fueland petrochemical industry among other applications. For example, themicroalgae Botryococcus braunii produces a high yield of up to 86% crudeweight of long chain hydrocarbons, a composition that is similar to highgrade crude oil. However, this microorganism has evolved in nature forsurvival in the wild rather than for rapid growth in a laboratory andwas previously believed to grow too slowly in its wild-type state toproduce a commercially viable source of hydrocarbons. Surprisingly, ithas been found that this species, which was known as an obligateautotroph, exhibits good growth when cultured in the dark with a fixedcarbon source.

Other microorganisms have naturally evolved for rapid growth in harshconditions in the wild (e.g., sewage processing pools) but do notproduce hydrocarbons in useful quantities.

The present application describes novel methods for culturing, as wellas methods for genetic modification of Botryococcus braunii and similarorganisms, to improve the rate and economics of growth in a cell cultureenvironment. The invention also provides methods of modifying othermicroorganisms that already have desired growth characteristics toacquire characteristics for producing large quantities of usefulhydrocarbons.

The resulting organisms having both high yields of useful hydrocarbonsand desired growth characteristics provide an alternative source ofhydrocarbons to the conventional petrochemical and oil industry. Thesehydrocarbons can be harvested from cell cultures and subjected tocatalysis to produce crude oil, gasoline, terpenoids, pharmaceuticalprecursors, rubber precursors and specialty chemical products. Somemicroalgae described herein can also be used for production ofhydrocarbons such as lutein.

Further, the invention provides genetically engineering strains ofmicroalgae with two or more exogenous genes. The first gene encodes atransporter of a fixed carbon source and the second gene encodes acarbohydrate modification enzyme. The resulting fermentable organismsproduce greater amounts of hydrocarbons per unit time as well ashydrocarbon compositions that contain greater energy content per unitweight than what has been obtainable by previously known methods ofgeological or biological hydrocarbon production. By providing theability to metabolize a fixed carbon source rather than only sunlightand carbon dioxide and the ability to steer metabolic carbon flux intohigh-energy content molecules at levels far greater than can be achievedin non-engineered organisms, the invention provides energy productionmethods far superior that those so far known. In other words, providingsaturating amounts of usable fixed carbon and inserting exogenous genesencoding enzymes that steer the fixed carbon into specificenergy-containing hydrocarbons allows for production of liquidhydrocarbons for transportation and other fuels at levels never beforepossible using microorganisms. Optionally, the insertion of the twoexogenous genes described above can be combined with the disruption ofpolysaccharide biosynthesis through directed and/or random mutagenesis,which steers ever greater carbon flux into hydrocarbon production.

Individually and in combination, trophic conversion, engineering toalter hydrocarbon production, and treatment with exogenous enzymes alterthe hydrocarbon composition produced by a microorganism. The alterationcan be a change in the amount of hydrocarbons produced, the amount ofone or more hydrocarbon species produced relative to other hydrocarbons,and/or the types of hydrocarbon species produced in the microorganism.

II Hydrocarbons

Hydrocarbons form a heterogeneous group of molecules of different sizes,shapes and/or lengths, and molecular weight. They are constructedprimarily or exclusively from hydrogen and carbon atoms wherein thecarbon atoms are covalently linked to form a linear, branched, cyclic,or partially cyclic backbone. The hydrogen atoms are attached to thecarbon atoms in the backbone. By structure, hydrocarbons arecharacterized by two main classes: aliphatic and aromatic. Aliphatichydrocarbons include alkanes, alkenes, and alkynes as well as theircyclic counterparts, which may be referred to as cyclic aliphatichydrocarbons. Generally, aliphatic compounds are open-chain instructure, such as linear, or are cyclic compounds that resemble theopen-chain structures.

Aromatic compounds are benzene and compounds that chemically resemblebenzene in behavior. Although aliphatic hydrocarbons generally undergoaddition (at locations of multiple bonds) and free-radical substitution(at other locations along the aliphatic chain), aromatic hydrocarbonstend to undergo heterolytic substitution. Aromatic compounds are alsocharacterized by their resonance structure.

Hydrocarbons can be used as an energy source based on the heat releasedon combustion. Examples include the combustion of methane, ethane,propane and butane as gases, as well as the combustion of largerhydrocarbons in the gaseous or liquid forms. Hydrocarbons have also beenutilized as the precursors, or subunits, for the production of polymerssuch as plastics.

Aliphatic alkanes are represented by the general formula C_(n)H_(2n)+2,which indicates the number of carbon and hydrogen atoms in an alkanemolecule. This formula can also be used as an example of the highestpossible hydrogen to carbon (hydrogen:carbon) ratio in a hydrocarbon ofa given carbon backbone structure. The higher the ratio, the more energyreleased upon combustion. Hydrocarbons with a hydrogen to carbon ratioabove about 2 are preferred for combustion. A cyclic aliphatichydrocarbon is represented by the general formula C_(n)H_(2n).

In an alkane, the carbon atoms in the carbon-carbon backbone are linkedvia carbon-carbon single bonds. Alkenes contain less hydrogen, on acarbon for carbon basis, than the alkanes. Thus, an alkene can beconverted to an alkane by addition of hydrogen. Conversely, an alkanecan be converted to an alkene by the loss of hydrogen. An alkenecontains less than the maximum amount of hydrogen on a carbon-carbonbackbone, so an alkene is referred to as an unsaturated hydrocarbon. Theunsaturated condition is present in the carbon-carbon backbone of analkene in the form of one or more carbon-carbon double bonds. Thesimplest alkene is ethylene.

Alkynes are another type of unsaturated hydrocarbon. In an alkyne, thecarbon-carbon backbone contains one or more carbon-carbon triple bonds.Like a double bond, the triple bond is highly reactive. The simplestalkyne is acetylene.

Terpenes are one type of hydrocarbon. Terpenes are compounds found inthe essential oils of various plants and among the hydrocarbons ofvarious microorganism. Terpenes are derived from isoprene, which may beconsidered the unit upon which a terpene is based. Isoprene has themolecular formula C₅H₈ and terpenes are represented by a formula formultiples of that. Thus (C₅H₈)_(n) where n is the number of linkedisoprene units is a formula which generally represents terpenestructure. This relationship between terpenes and isoprene is alsoreferred to as the isoprene rule or the C₅ rule. In some terpenes, theindividual isoprene units may be linked “head to tail” to form linearchains. In other terpenes, the isoprene units are arranged to formrings.

Terpenes can be modified chemically to form terpenoids for example byoxidation or rearrangement of the carbon backbone of terpene. Examplesof terpenes include hemiterpenes, with one isoprene unit whereoxygen-containing derivatives like prenol and isovaleric acid arehemiterpenoids; monoterpenes, with two isoprene units and represented bythe formula C₁₀H₁₆; sesquiterpenes, with three isoprene units andrepresented by the formula C₁₅H₂₄; diterpenes, with four isoprene unitsand represented by the formula C₂₀H₃₂; sesterterpenes, with 25 carbonsand five isoprene units; triterpenes, with six isoprene units andrepresented by the formula C₃₀H₄₈; tetraterpenes, with eight isopreneunits and represented by the formula C₄₀H₅₆; and polyterpenes, with longchains of many isoprene units.

Examples of diterpenes include cembrene and taxadiene. Diterpenes arealso the basis for biological compounds such as retinol, retinal, andphytol. A non-limiting example of a triterpene is the linear triterpenesqualene. Examples of tetraterpenes include the acyclic lycopene, themonocyclic gamma-carotene, and the bicyclic alpha- and beta-carotenes. Arepresentative example of a polyterpene is rubber, consisting ofpolyisoprene in which the double bonds are cis.

Lipids are another type of hydrocarbon containing molecule. Examples oflipids include fatty acids (saturated and unsaturated); glycerides orglycerolipids (such as monoglycerides, diglycerides, triglycerides orneutral fats, and phosphoglycerides or glycerophospholipids);nonglycerides (sphingolipids, sterol lipids including cholesterol andsteroid hormones, prenol lipids including terpenoids, waxes, andpolyketides); and complex lipid derivatives (sugar-linked lipids, orglycolipids, and protein-linked lipids). In some embodiments, the lipidis a phospholipid, such as farnesyl diphosphate.

Long chain hydrocarbons are particularly useful for the petrochemicalindustry. In some embodiments, a long chain hydrocarbon contains atleast about 8, at least about 10, at least about 12, at least about 14,at least about 16, at least about 18, at least about 20, at least about22, at least about 24, at least about 26, at least about 28, at leastabout 30, at least about 32, or at least about 34 carbon atoms or more.Other embodiments include hydrocarbons, such as the above long chainhydrocarbons, that are alkanes (with no carbon-carbon double or triplebonds); that are linear (not cyclic); and/or that have little or nobranching in their structures. Hydrocarbons with a hydrogen: carbonratio above about 2, up to 4 in the case of methane, are included asembodiments herein.

Different hydrocarbon chain lengths all have progressively higherboiling points, so they can all be separated by distillation. Forexample, crude oil is separated into various fractions by heating andseparating different chain lengths when they vaporize at differenttemperatures. Each different chain length has a different property thatmakes it useful in a different way. Petroleum gas used for heating,cooking, and making plastics contains small alkanes (1 to 4 carbonatoms), e.g., methane, ethane, propane, butane having a boiling rangeless than 40 degrees. Naphtha or ligroin are intermediates furtherprocessed to make gasoline. They contain 5 to 9 carbon atom alkanes andhave a boiling range of 60 to 100 degrees Celsius. Gasoline has a mix ofalkanes and cycloalkanes of 5 to 12 carbon atoms and a boiling range of40 to 205 degrees Celsius. Kerosene, which is the fuel for jet enginesand tractors and a starting material for making other products is a mixof alkanes of 10 to 18 carbons and aromatics and has a boiling range of175 to 325 degrees Celsius. Gas oil or diesel distillate, which is usedfor diesel fuel and heating oil; starting material for making otherproducts contains alkanes containing of 12 or more carbon atoms and hasa boiling range of 250 to 350 degrees Celsius. Lubricating oil, which isused for motor oil, grease, and other lubricants contains long chains of20 to 50 carbon atoms and includes alkanes, cycloalkanes, aromatics, andhas a boiling range of 300 to 370 degrees Celsius. Heavy gas or fueloil, which is used for industrial fuel and as a starting material formaking other products contains long chain of 20 to 70 carbon atomsincluding alkanes, cycloalkanes, aromatics and has a boiling range of370 to 600 degrees Celsius. Residuals include coke, asphalt, tar, waxes;starting material for making other products, which are multiple-ringedcompounds with 70 or more carbon atoms and a boiling range greater than600 degrees Celsius. It is an object of the invention to providegenetically engineered microorganisms, particularly microalgae, thatproduce one or more species of hydrocarbons disclosed in this and theprevious paragraph, as well as precursors to these molecules that can beput through refining and/or catalysis and/or cracking to produce themolecules disclosed in this and the previous paragraph.

III Suitable Microorganisms

Desired microorganisms for use in the invention produce high yields ofhydrocarbons, and/or grow rapidly on a fixed carbon source. Any speciesof organism that produces hydrocarbons can be used, althoughmicroorganisms that naturally produce high levels of hydrocarbons arepreferred. Production of hydrocarbons by microorganisms is reviewed byMetzger et al. Appl Microbiol Biotechnol (2005) 66: 486-496. Nonlimitingexamples of photosynthetic microorganisms, listed as both genus andspecies, that can be used can be found in Table I.

TABLE I Achnanthes orientalis Agmenellum Amphiprora hyaline Amphoracoffeiformis Amphora coffeiformis linea Amphora coffeiformis punctataAmphora coffeiformis taylori Amphora coffeiformis tenuis Amphoradelicatissima Amphora delicatissima capitata Amphora sp. AnabaenaAnkistrodesmus Ankistrodesmus falcatus Boekelovia hooglandii Borodinellasp. Botryococcus braunii Botryococcus sudeticus Carteria Chaetocerosgracilis Chaetoceros muelleri Chaetoceros muelleri subsalsum Chaetocerossp. Chlorella ellipsoidea Chlorella salina Chlorella sp. Chlorococcuminfusionum Chlorococcum sp. Chlorogonium Chroomonas sp. Chrysosphaerasp. Cricosphaera sp. Cryptomonas sp. Cyclotella cryptica Cyclotellameneghiniana Cyclotella sp. Dunaliella sp. Dunaliella bardawilDunaliella bioculata Dunaliella granulata Dunaliella maritima Dunaliellaminuta Dunaliella parva Dunaliella peircei Dunaliella primolectaDunaliella salina Dunaliella terricola Dunaliella tertiolecta Dunaliellaviridis Dunaliella tertiolecta Eremosphaera viridis Eremosphaera sp.Ellipsoidon sp. Euglena Franceia sp. Fragilaria crotonensis Fragilariasp. Gleocapsa sp. Gloeothamnion sp. Hymenomonas sp. Isochrysis aff.galbana Isochrysis galbana Lepocinclis Monoraphidium minutumMonoraphidium sp. Nannochloris sp. Nannochloropsis salinaNannochloropsis sp. Navicula acceptata Navicula biskanterae Naviculapseudotenelloides Navicula saprophila Navicula sp. Nephrochloris sp.Nephroselmis sp. Nitschia communis Nitzschia alexandrina Nitzschiacommunis Nitzschia dissipata Nitzschia frustulum Nitzschia hantzschianaNitzschia inconspicua Nitzschia intermedia Nitzschia microcephalaNitzschia pusilla Nitzschia pusilla elliptica Nitzschia pusillamonoensis Nitzschia quadrangula Nitzschia sp. Ochromonas sp. Oocystisparva Oocystis pusilla Oocystis sp. Oscillatoria limnetica Oscillatoriasp. Oscillatoria subbrevis Pascheria acidophila Pavlova sp. PhagusPhormidium Platymonas sp. Pleurochrysis carterae Pleurochrysis dentatePleurochrysis sp. Pyramimonas sp. Pyrobotrys Sarcinoid chrysophyteSpirogyra Stichococcus sp. Synechococcus sp. Tetraedron, Tetraselmis sp.Tetraselmis suecica Thalassiosira weissflogii

Botryococcus, particularly Botryococcus braunii, is a preferredmicroorganism because of its high yield and composition of hydrocarbons,particularly long chain hydrocarbons. Pyrobotrys, Phormidium,Agmenellum, Carteria, Lepocinclis, Pyrobotrys, Nitzschia, Lepocinclis,Anabaena, Euglena, Spirogyra, Chlorococcum, Tetraedron, Oscillatoria,Phagus and Chlorogonium are also useful because of their capacity togrow rapidly in the harsh conditions of wastewater and sewage treatmentpools and their ability to recycle waste products.

Non-photosynthetic microorganisms can also be used to producehydrocarbons, such as E. coli, Bacillus, Saccromyces, and other microbesthat are preferably amenable to genetic engineering. For example, E.coli has been used to manufacture hydrocarbons such as carotenoids (seefor example Appl Microbiol Biotechnol. 2006 Apr. 14, Characterization ofbacterial beta-carotene 3,3′-hydroxylases, CrtZ, and P450 in astaxanthinbiosynthetic pathway and adonirubin production by gene combination inEscherichia coli, Choi S K et al.). Such microorganisms include obligateheterotrophs which naturally produce hydrocarbons. Alternatively,heterotrophs can be recombinantly modified to enhance production of ahydrocarbon. For example, heterotrophs can be transformed with a nucleicacid sequence that encodes a beta carotene hydroxylase.

IV Trophic Conversion

Trophic conversion refers to the process of recombinantly inserting anucleic acid sequence into a photoautotrophic cell such that it gainsthe capability of relying upon a fixed carbon source (see Zaslayskaia etal. Science (2001)292:2073-2075). Some microorganisms includingBotryococcus braunii are photoautotrophic organisms, meaning that theseorganisms in their wild-type state rely upon light as an energy sourceand carbon dioxide as a carbon source for cellular activities andfunctions. An obligate photoautotroph is unable to utilize a fixedcarbon source in its environment as an energy source. This is incontrast to heterotrophic organisms which can utilize a fixed carbonsource (such as glucose) as an energy source. Mixotrophic organisms arecapable of deriving metabolic energy both from photosynthesis and fromexternal energy sources. Some microalgae such as Chlorella can growheterotrophically (in the dark on a fixed carbon source),photoautotrophically (using only light as an energy source), ormixotrophically (in the presence of both light and a fixed carbonsource). In the presence of light, Botryococcus braunii growth can beinhibited by the inclusion of carbohydrates in the culture medium.However, the inventors have discovered that this species can utilizefixed carbon sources, including carbohydrates, and exhibits good growthcharacteristics when cultured in the absence of light.

In many instances, the inability of photoautotrophs to use a fixedcarbon source arises from lack of a transporter to take up the sourceinto the cell. Many cells lacking such a transporter can neverthelessmetabolize a fixed carbon source once it is taken up into the cell.

Photoautotrophs can be converted to heterophophic or mixotrophicorganisms by genetic transformation. This added function confers theability for the cell to grow and propagate in the absence of light andphotosynthesis (such as in the dark) but in the presence of the fixedcarbon source. The nucleic acid sequence can be a gene encoding amembrane-associated transporter that transports a fixed carbon source,such as glucose, into the cell. In some instances two genes are requiredto trophically convert a photoautotroph: a first gene encoding atransporter that transports a fixed carbon source into the cell, and asecond gene encoding an enzyme with hexokinase activity thatphosphorylates a hexose molecule such as glucose. Some organisms requirean exogenous hexokinase gene to convert a fixed carbon source into aphosphorylated form that can be utilized by the endogenous metabolicpathways of the cell. Many obligate photoautotrophs contain endogenousgenes encoding enzymes with hexokinase activity. Whether a hexokinasegene is required for trophic conversion can be determined byradiolabeling a fixed carbon source such as glucose and exposing cellsexpressing a transporter to the radiolabeled glucose. Cells thattransport the labeled glucose but are not capable of growth in theabsence of light can be trophically converted by being transformed by asecond gene encoding a hexokinase, followed by selection in the dark onmedia containing glucose. The gene encoding the transporter or othergene is in operable linkage to a promoter active in microalgae andoptionally other regulatory sequences, such as introns and enhancers,that allow or facilitate expression. Trophic conversion providesadvantages such as increased, or faster, growth rates, shorter growthtimes, and very high cell densities in culture. The need for lightenergy is reduced or eliminated because the cells may grow and producecellular products, including hydrocarbons, in the presence of fixedcarbon material as the energy source.

Preferred cells for trophic conversion include Pyrobotrys, Phormidium,Agmenellum, Carteria, Lepocinclis, Pyrobotrys, Nitzschia, Lepocinclis,Anabaena, Euglena, Spirogyra, Chlorococcum, Tetraedron, Oscillatoria,Phagus, Chlorogonium, Dunaliella or Botryococcus cells. Optionally, suchcells are transformed with one or more transporters having substratespecificities that allow transport of multiple carbon sources, such asthose found in municipal wastewater and/or secondarily treated sewage.Examples of such multisubstrate transporters are described herein in thesequence listing. Other such cells can be produced by chemical ornonchemical mutagenesis of natural cells or cells transformed with atransporter. Transformed cells are selected on a carbon source in theabsence of light. The selection can be, for example, on about 0.1% orabout 1% glucose, or another fixed carbon source, in the dark.Alternatively, the microalgae can be transformed with a vectorcontaining both an antibiotic resistance gene, such as a gene encodingresistance to the antibiotic zeocin, and a carbohydrate transporter withselection for antibiotic resistance. New strains exhibiting antibioticresistance can be then tested for the ability to grow in the dark in thepresence of a fixed carbon source that is transported by thecarbohydrate transporter. Carbon sources suitable for use in theinvention can be found below in Table II. A preferred carbon source isdepolymerized cellulose in the form of a mixture of xylose and glucose,optionally including arabinose, as described for example in Wyman etal., Comparative sugar recovery data from laboratory scale applicationof leading pretreatment technologies to corn stover, Bioresour Technol.2005 December; 96(18):2026-32; Gusakov et al., Design of highlyefficient cellulase mixtures for enzymatic hydrolysis of cellulose,Biotechnol Bioeng 2007 Jan. 12; Jeoh et al., Cellulase digestibility ofpretreated biomass is limited by cellulose accessibility, BiotechnolBioeng. 2007 Mar. 2; Lawford et al., Performance testing of Zymomonasmobilis metabolically engineered for cofermentation of glucose, xylose,and arabinose, Appl Biochem Biotechnol. 2002 Spring; 98-100:429-48.

TABLE II 2,3-Butanediol 2-Aminoethanol 2′-Deoxy Adenosine 3-MethylGlucose Acetic Acid Adenosine Adenosine-5′-Monophosphate AdonitolAmygdalin Arbutin Bromosuccinic Acid Cis-Aconitic Acid Citric AcidD,L-Carnitine D,L-Lactic Acid D,L-α-Glycerol Phosphate D-AlanineD-Arabitol D-Cellobiose Dextrin D-Fructose D-Fructose-6-PhosphateD-Galactonic Acid Lactone D-Galactose D-Galacturonic Acid D-GluconicAcid D-Glucosaminic Acid D-Glucose-6-Phosphate D-Glucuronic AcidD-Lactic Acid Methyl Ester D-L-α-Glycerol Phosphate D-Malic AcidD-Mannitol D-Mannose D-Melezitose D-Melibiose D-Psicose D-RaffinoseD-Ribose D-Saccharic Acid D-Serine D-Sorbitol D-Tagatose D-TrehaloseD-Xylose Formic Acid Gentiobiose Glucuronamide Glycerol GlycogenGlycyl-LAspartic Acid Glycyl-LGlutamic Acid Hydroxy-LProlinei-Erythritol Inosine Inulin Itaconic Acid Lactamide LactuloseL-Alaninamide L-Alanine L-Alanylglycine L-Alanyl-Glycine L-ArabinoseL-Asparagine L-Aspartic Acid L-Fucose L-Glutamic Acid L-HistidineL-Lactic Acid L-Leucine L-Malic Acid L-Ornithine L-PhenylalanineL-Proline L-Pyroglutamic Acid L-Rhamnose L-Serine L-Threonine MalonicAcid Maltose Maltotriose Mannan m-Inositol N-Acetyl-DGalactosamineN-Acetyl-DGlucosamine N-Acetyl-LGlutamic Acid N-Acetyl-β-DMannosaminePalatinose Phenyethylamine p-Hydroxy-Phenylacetic Acid Propionic AcidPutrescine Pyruvic Acid Pyruvic Acid Methyl Ester Quinic Acid SalicinSebacic Acid Sedoheptulosan Stachyose Succinamic Acid Succinic AcidSuccinic Acid Mono-Methyl-Ester Sucrose ThymidineThymidine-5′-Monophosphate Turanose Tween 40 Tween 80 UridineUridine-5′-Monophosphate Urocanic Acid Water Xylitol α-Cyclodextrinα-D-Glucose α-D-Glucose-1-Phosphate α-D-Lactose α-Hydroxybutyric Acidα-Keto Butyric Acid α-Keto Glutaric Acid α-Keto Valeric Acidα-Ketoglutaric Acid α-Ketovaleric Acid α-Methyl-DGalactosideα-Methyl-DGlucoside α-Methyl-DMannoside β-Cyclodextrin β-HydroxybutyricAcid β-Methyl-DGalactoside β-Methyl-D-Glucoside γ-Amino Butyric Acidγ-Hydroxybutyric Acid(2-amino-3,4-dihydroxy-5-hydroxymethyl-1-cyclohexyl)glucopyranoside(3,4-disinapoyl)fructofuranosyl-(6-sinapoyl)glucopyranoside(3-sinapoyl)fructofuranosyl-(6-sinapoyl)glucopyranoside 1 reference1,10-di-O-(2-acetamido-2-deoxyglucopyranosyl)-2-azi-1,10-decanediol1,3-mannosylmannose 1,6-anhydrolactose 1,6-anhydrolactose hexaacetate1,6-dichlorosucrose 1-chlorosucrose 1-desoxy-1-glycinomaltose1-O-alpha-2-acetamido-2-deoxygalactopyranosyl-inositol1-O-methyl-di-N-trifluoroacetyl-beta-chitobioside 1-propyl-4-O-betagalactopyranosyl-alpha galactopyranoside2-(acetylamino)-4-O-(2-(acetylamino)-2-deoxy-4-O-sulfogalactopyranosyl)-2-deoxyglucose 2-(trimethylsilyl)ethyl lactoside2,1′,3′,4′,6′-penta-O-acetylsucrose2,2′-O-(2,2′-diacetamido-2,3,2′,3′-tetradeoxy-6,6′-di-O-(2-tetradecylhexadecanoyl)-alpha,alpha′-trehalose-3,3′-diyl)bis(N-lactoyl-alanyl-isoglutamine) 2,3,6,2′,3′,4′,6′-hepta-O-acetylcellobiose2,3′-anhydrosucrose2,3-di-O-phytanyl-1-O-(mannopyranosyl-(2-sulfate)-(1-2)-glucopyranosyl)-sn-glycerol 2,3-epoxypropylO-galactopyranosyl(1-6)galactopyranoside2,3-isoprolylideneerthrofuranosyl 2,3-O-isopropylideneerythrofuranoside2′,4′-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside2,5-anhydromannitol iduronate 2,6-sialyllactose2-acetamido-2,4-dideoxy-4-fluoro-3-O-galactopyranosylglucopyranose2-acetamido-2-deoxy-3-O-(gluco-4-enepyranosyluronic acid)glucose2-acetamido-2-deoxy-3-O-rhamnopyranosylglucose2-acetamido-2-deoxy-6-O-beta galactopyranosylgalactopyranose2-acetamido-2-deoxyglucosylgalactitol2-acetamido-3-O-(3-acetamido-3,6-dideoxy-beta-glucopyranosyl)-2-deoxy-galactopyranose2-amino-6-O-(2-amino-2-deoxy-glucopyranosyl)-2-deoxyglucose2-azido-2-deoxymannopyranosyl-(1,4)-rhamnopyranose2-deoxy-6-O-(2,3-dideoxy-4,6-O-isopropylidene-2,3-(N-tosylepimino)mannopyranosyl)-4,5-O-isopropylidene-1,3-di-N-tosylstreptamine 2-deoxymaltose 2-iodobenzyl-1-thiocellobioside2-N-(4-benzoyl)benzoyl-1,3-bis(mannos-4-yloxy)-2-propylamine2-nitrophenyl-2-acetamido-2-deoxy-6-O-beta galactopyranosyl-alphagalactopyranoside 2-O-(glucopyranosyluronic acid)xylose2-O-glucopyranosylribitol-1-phosphate2-O-glucopyranosylribitol-4′-phosphate2-O-rhamnopyranosyl-rhamnopyranosyl-3-hydroxyldecanoyl-3-hydroxydecanoate 2-O-talopyranosylmannopyranoside 2-thiokojibiose2-thiosophorose 3,3′-neotrehalosadiamine3,6,3′,6′-dianhydro(galactopyranosylgalactopyranoside)3,6-di-O-methyl-beta-glucopyranosyl-(1-4)-2,3-di-O-methyl-alpha-rhamnopyranose3-amino-3-deoxyaltropyranosyl-3-amino-3-deoxyaltropyranoside3-deoxy-3-fluorosucrose 3-deoxy-5-O-rhamnopyranosyl-2-octulopyranosonate3-deoxyoctulosonic acid-(alpha-2-4)-3-deoxyoctulosonic acid3-deoxysucrose 3-ketolactose 3-ketosucrose 3-ketotrehalose3-methyllactose3-O-(2-acetamido-6-O-(N-acetylneuraminyl)-2-deoxygalactosyl)serine3-O-(glucopyranosyluronic acid)galactopyranose3-O-beta-glucuronosylgalactose3-O-fucopyranosyl-2-acetamido-2-deoxyglucopyranose3′-O-galactopyranosyl-1-4-O-galactopyranosylcytarabine3-O-galactosylarabinose 3-O-talopyranosylmannopyranoside 3-trehalosamine4-(trifluoroacetamido)phenyl-2-acetamido-2-deoxy-4-O-beta-mannopyranosyl-beta-glucopyranoside4,4′,6,6′-tetrachloro-4,4′,6,6′-tetradeoxygalactotrehalose4,6,4′,6′-dianhydro(galactopyranosylgalactopyranoside)4,6-dideoxysucrose 4,6-O-(1-ethoxy-2-propenylidene)sucrose hexaacetate4-chloro-4-deoxy-alpha-galactopyranosyl3,4-anhydro-1,6-dichloro-1,6-dideoxy-beta-lyxo-hexulofuranoside 4-glucopyranosylmannose4-methylumbelliferylcellobioside 4-nitrophenyl2-fucopyranosyl-fucopyranoside 4-nitrophenyl2-O-alpha-D-galactopyranosyl-alpha-D-mannopyranoside 4-nitrophenyl2-O-alpha-D-glucopyranosyl-alpha-D-mannopyranoside 4-nitrophenyl2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside 4-nitrophenyl6-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside4-nitrophenyl-2-acetamido-2-deoxy-6-O-beta-D-galactopyranosyl-beta-D-glucopyranoside4-O-(2-acetamido-2-deoxy-beta-glucopyranosyl)ribitol4-O-(2-amino-2-deoxy-alpha-glucopyranosyl)-3-deoxy-manno-2- octulosonicacid 4-O-(glucopyranosyluronic acid)xylose4-O-acetyl-alpha-N-acetylneuraminyl-(2-3)-lactose4-O-alpha-D-galactopyranosyl-D-galactose4-O-galactopyranosyl-3,6-anhydrogalactose dimethylacetal4-O-galactopyranosylxylose 4-O-mannopyranosyl-2-acetamido-2-deoxyglucose4-thioxylobiose 4-trehalosamine 4-trifluoroacetamidophenyl2-acetamido-4-O-(2-acetamido-2-deoxyglucopyranosyl)-2-deoxymannopyranosiduronic acid5-bromoindoxyl-beta-cellobioside5′-O-(fructofuranosyl-2-1-fructofuranosyl)pyridoxine5-O-beta-galactofuranosyl-galactofuranose 6 beta-galactinol6(2)-thiopanose 6,6′-di-O-corynomycoloyl-alpha-mannopyranosyl-alpha-mannopyranoside 6,6-di-O-maltosyl-beta-cyclodextrin6,6′-di-O-mycoloyl-alpha-mannopyranosyl-alpha-mannopyranoside6-chloro-6-deoxysucrose 6-deoxy-6-fluorosucrose6-deoxy-alpha-gluco-pyranosiduronic acid 6-deoxy-gluco-heptopyranosyl6-deoxy-gluco-heptopyranoside 6-deoxysucrose6-O-decanoyl-3,4-di-O-isobutyrylsucrose6-O-galactopyranosyl-2-acetamido-2-deoxygalactose6-O-galactopyranosylgalactose 6-O-heptopyranosylglucopyranose6-thiosucrose 7-O-(2-amino-2-deoxyglucopyranosyl)heptose8-methoxycarbonyloctyl-3-O-glucopyranosyl-mannopyranoside8-O-(4-amino-4-deoxyarabinopyranosyl)-3-deoxyoctulosonic acidallolactose allosucrose allyl 6-O-(3-deoxyoct-2-ulopyranosylonicacid)-(1-6)-2-deoxy-2-(3- hydroxytetradecanamido)glucopyranoside4-phosphate alpha-(2-9)-disialic acid alpha,alpha-trehalose6,6′-diphosphate alpha-glucopyranosyl alpha-xylopyranosidealpha-maltosyl fluoride aprosulate benzyl2-acetamido-2-deoxy-3-O-(2-O-methyl-beta-galactosyl)-beta-glucopyranoside benzyl 2-acetamido-2-deoxy-3-O-beta fucopyranosyl-alpha-galactopyranoside benzyl 2-acetamido-6-O-(2-acetamido-2,4-dideoxy-4-fluoroglucopyranosyl)-2-deoxygalactopyranoside benzyl gentiobiosidebeta-D-galactosyl(1-3)-4-nitrophenyl-N-acetyl-alpha-D-galactosaminebeta-methylmelibiose calcium sucrose phosphate camiglibose cellobialcellobionic acid cellobionolactone Cellobiose cellobiose octaacetatecellobiosyl bromide heptaacetate Celsior chitobiose chondrosineCristolax deuterated methyl beta-mannobioside dextrin maltoseD-glucopyranose, O-D-glucopyranosyl Dietary Sucrose difructose anhydrideI difructose anhydride III difructose anhydride IV digalacturonic acidDT 5461 ediol epilactose epsilon-N-1-(1-deoxylactulosyl)lysine feruloylarabinobiose floridoside fructofuranosyl-(2-6)-glucopyranoside FZ 560galactosyl-(1-3)galactose garamine gentiobiose geranyl6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside geranyl6-O-xylopyranosyl-glucopyranoside glucosaminyl-1,6-inositol-1,2-cyclicmonophosphate glucose glucosyl (1-4) N-acetylglucosamineglucuronosyl(1-4)-rhamnose heptosyl-2-keto-3-deoxyoctonate inulobioseIsomaltose isomaltulose isoprimeverose kojibiose lactobionic acidlacto-N-biose II Lactose lactosylurea Lactulose laminaribioselepidimoide leucrose levanbiose lucidin 3-O-beta-primveroside LW 10121LW 10125 LW 10244 maltal maltitol Maltose maltose hexastearatemaltose-maleimide maltosylnitromethane heptaacetatemaltosyltriethoxycholesterol maltotetraose Malun 25 mannosucrosemannosyl-(1-4)-N-acetylglucosaminyl-(1-N)-ureamannosyl(2)-N-acetyl(2)-glucose melibionic acid Melibiose melibiouronicacid methyl 2-acetamido-2-deoxy-3-O-(alpha-idopyranosyluronic acid)-4-O-sulfo-beta-galactopyranoside methyl2-acetamido-2-deoxy-3-O-(beta-glucopyranosyluronic acid)-4-O-sulfo-beta-galactopyranoside methyl2-acetamido-2-deoxy-3-O-glucopyranosyluronoylglucopyranoside methyl2-O-alpha-rhamnopyranosyl-beta-galactopyranoside methyl2-O-beta-rhamnopyranosyl-beta-galactopyranoside methyl2-O-fucopyranosylfucopyranoside 3 sulfate methyl2-O-mannopyranosylmannopyranoside methyl2-O-mannopyranosyl-rhamnopyranoside methyl 3-O-(2-acetamido-2-deoxy-6-thioglucopyranosyl)galactopyranoside methyl3-O-galactopyranosylmannopyranoside methyl3-O-mannopyranosylmannopyranoside methyl3-O-mannopyranosyltalopyranoside methyl 3-O-talopyranosyltalopyranosidemethyl 4-O-(6-deoxy-manno-heptopyranosyl)galactopyranoside methyl6-O-acetyl-3-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzoylgalactopyranosyl)-2-deoxy-2-phthalimidoglucopyranoside methyl6-O-mannopyranosylmannopyranoside methyl beta-xylobioside methylfucopyranosyl(1-4)-2-acetamido-2-deoxyglucopyranoside methyllaminarabioside methylO-(3-deoxy-3-fluorogalactopyranosyl)(1-6)galactopyranosidemethyl-2-acetamido-2-deoxyglucopyranosyl-1-4-glucopyranosiduronic acidmethyl-2-O-fucopyranosylfucopyranoside 4-sulfate MK 458N(1)-2-carboxy-4,6-dinitrophenyl-N(6)-lactobionoyl-1,6-hexanediamineN-(2,4-dinitro-5-fluorophenyl)-1,2-bis(mannos-4′-yloxy)propyl-2-amineN-(2′-mercaptoethyl)lactamineN-(2-nitro-4-azophenyl)-1,3-bis(mannos-4′-yloxy)propyl-2-amineN-(4-azidosalicylamide)-1,2-bis(mannos-4′-yloxy)propyl-2-amineN,N-diacetylchitobiose N-acetylchondrosine N-acetyldermosineN-acetylgalactosaminyl-(1-4)-galactoseN-acetylgalactosaminyl-(1-4)-glucoseN-acetylgalactosaminyl-1-4-N-acetylglucosamineN-acetylgalactosaminyl-1-4-N-acetylglucosamineN-acetylgalactosaminyl-alpha(1-3)galactoseN-acetylglucosamine-N-acetylmuramyl-alanyl-isoglutaminyl-alanyl-glycerol dipalmitoyl N-acetylglucosaminyl beta(1-6)N-acetylgalactosamineN-acetylglucosaminyl-1-2-mannopyranose N-acetylhyalobiuronic acidN-acetylneuraminoyllactose N-acetylneuraminoyllactose sulfate esterN-acetylneuraminyl-(2-3)-galactose N-acetylneuraminyl-(2-6)-galactoseN-benzyl-4-O-(beta-galactopyranosyl)glucamine-N-carbodithioateneoagarobiose N-formylkansosaminyl-(1-3)-2-O-methylrhamnopyranoseO-((Nalpha)-acetylglucosamine 6-sulfate)-(1-3)-idonic acidO-(4-O-feruloyl-alpha-xylopyranosyl)-(1-6)-glucopyranoseO-(alpha-idopyranosyluronic acid)-(1-3)-2,5-anhydroalditol-4-sulfateO-(glucuronic acid 2-sulfate)-(1--3)-O-(2,5)-andydrotalitol 6-sulfateO-(glucuronic acid 2-sulfate)-(1--4)-O-(2,5)-anhydromannitol 6-sulfateO-alpha-glucopyranosyluronate-(1-2)-galactoseO-beta-galactopyranosyl-(1-4)-O-beta-xylopyranosyl-(1-0)-serine octylmaltopyranoside O-demethylpaulomycin A O-demethylpaulomycin BO-methyl-di-N-acetyl beta-chitobioside Palatinit paldimycin paulomenol Apaulomenol B paulomycin A paulomycin A2 paulomycin B paulomycin Cpaulomycin D paulomycin E paulomycin F phenyl2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside phenylO-(2,3,4,6-tetra-O-acetylgalactopyranosyl)-(1-3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-1-thioglucopyranosidepoly-N-4-vinylbenzyllactonamide pseudo-cellobiose pseudo-maltoserhamnopyranosyl-(1-2)-rhamnopyranoside-(1-methyl ether) rhoifolinruberythric acid S-3105 senfolomycin A senfolomycin B solabiose SS 554streptobiosamine Sucralfate Sucrose sucrose acetate isobutyrate sucrosecaproate sucrose distearate sucrose monolaurate sucrose monopalmitatesucrose monostearate sucrose myristate sucrose octaacetate sucroseoctabenzoic acid sucrose octaisobutyrate sucrose octasulfate sucrosepolyester sucrose sulfate swertiamacroside T-1266 tangshenoside Itetrahydro-2-((tetrahydro-2-furanyl)oxy)-2H-pyran thionigerose Trehalosetrehalose 2-sulfate trehalose 6,6′-dipalmitate trehalose-6-phosphatetrehalulose trehazolin trichlorosucrose tunicamine turanose U 77802 U77803 xylobiose xylose-glucose xylosucrose

In certain embodiments, Botryococcus braunii is cultured using glucose,mannose, galactose, fructose, or glycerol, or a combination thereof, asthe fixed carbon source. Optionally urea can be added to the media aswell. Preferably, such cultures are grown in the dark, i.e., onceculturing is begun, no light is permitted to strike the microalgae.

In other embodiments, other components are added to the media including,but not limited to, dextrin, malt extract, traders yeast, corn meal,corn steep powder, whole dead yeast, casein type M, casein type B,tomato paste, molasses, soy hydrolysate, soy flour, corn starch andmaltose.

An exemplary vector design for expression of a gene in microalgaecontains a first gene encoding a transporter in operable linkage with apromoter active in microalgae. Alternatively, if the vector does notcontain a promoter in operable linkage with the first gene, the firstgene can be transformed into the cells such that it becomes operablylinked to an endogenous promoter at the point of vector integration. Thepromoterless method of transformation has been proven to work inmicroalgae (see for example Plant Journal 14:4, (1998), pp. 441-447),though the frequency of transformation is lower using this method thanwhen using a promoter active in microalgae in operable linkage with thefirst gene. The vector can also contain a second gene encodes a proteinthat imparts resistance to an antibiotic or herbicide. Optionally,either gene is followed by a 3′ untranslated sequence containing apolyadenylation signal. Expression cassettes encoding the two genes canbe physically linked in the vector or on separate vectors.Co-transformation of microalgae can also be used, in which distinctvector molecules are simultaneously used to transform cells (see forexample Protist. 2004 December; 155(4):381-93). The transformed cellscan be optionally selected based upon the ability to grow in thepresence of the antibiotic or other selectable marker under conditionsin which cells lacking the resistance cassette would not grow, such asin the dark. Including of the selectable marker is optional becauseobligate photoautotrophism provides an alternative means to select forexpression of a sugar transporter. Correct expression and functionalityof the transporter as well as the ability to metabolize transportedfixed carbon is the selectable phenotype when cells are grown in theabsence of light for photosynthesis.

DNA encoding the transporter and resistance gene is preferablycodon-optimized cDNAs. Methods of recoding genes for expression inmicroalgae are described in US Patent Application 20040209256.Additional information is available at the web addresswww.kazusa.or.jp/codon.

Many promoters in expression vectors are active in microalgae, includingboth promoters that are endogenous to the algae being transformed algaeas well as promoters that are not endogenous to the algae beingtransformed (i.e., promoters from other algae, promoters from higherplants, and promoters from plant viruses or algae viruses). Exogenousand/or endogenous promoters that are active in microalgae, andantibiotic resistance genes functional in microalgae are described bye.g., Curr Microbiol. 1997 December; 35(6):356-62 (Chlorella vulgaris);Mar Biotechnol (NY). 2002 January; 4(1):63-73 (Chlorella ellipsoidea);Mol Gen Genet. 1996 Oct. 16; 252(5):572-9 (Phaeodactylum tricornutum);Plant Mol. Biol. 1996 April; 31(1):1-12 (Volvox carteri); Proc Natl AcadSci USA. 1994 Nov. 22; 91(24):11562-6 (Volvox carteri); Falciatore A,Casotti R, Leblanc C, Abrescia C, Bowler C, PMID: 10383998, 1999 May;1(3):239-251 (Laboratory of Molecular Plant Biology, Stazione Zoologica,Villa Comunale, 1-80121 Naples, Italy) (Phaeodactylum tricornutum andThalassiosira weissflogii); Plant Physiol. 2002 May; 129(1):7-12.(Porphyridium sp.); Proc Natl Acad Sci USA. 2003 Jan. 21; 100(2):438-42.(Chlamydomonas reinhardtii); Proc Natl Acad Sci USA. 1990 February;87(3):1228-32. (Chlamydomonas reinhardtii); Nucleic Acids Res. 1992 Jun.25; 20(12):2959-65; Mar Biotechnol (NY). 2002 January; 4(1):63-73(Chlorella); Biochem Mol Biol Int. 1995 August; 36(5):1025-35(Chlamydomonas reinhardtii); J. Microbiol. 2005 August; 43(4):361-5(Dunaliella); Yi Chuan Xue Bao. 2005 April; 32(4):424-33 (Dunaliella);Mar Biotechnol (NY). 1999 May; 1(3):239-251. (Thalassiosira andPhaedactylum); Koksharova, Appl Microbiol Biotechnol 2002 February;58(2):123-37 (various species); Mol Genet Genomics. 2004 February;271(1):50-9 (Thermosynechococcus elongates); J. Bacteriol. (2000), 182,211-215; FEMS Microbiol Lett. 2003 Apr. 25; 221(2):155-9; Plant Physiol.1994 June; 105(2):635-41; Plant Mol. Biol. 1995 December; 29(5):897-907(Synechococcus PCC 7942); Mar Pollut Bull. 2002; 45(1-12):163-7(Anabaena PCC 7120); Proc Natl Acad Sci USA. 1984 March; 81(5):1561-5(Anabaena (various strains)); Proc Natl Acad Sci USA. 2001 Mar. 27;98(7):4243-8 (Synechocystis); Wirth, Mol Gen Genet. 1989 March;216(1):175-7 (various species); Mol Microbiol, 2002 June; 44(6):1517-31and Plasmid, 1993 September; 30(2):90-105 (Fremyella diplosiphon); Hallet al. (1993) Gene 124: 75-81 (Chlamydomonas reinhardtii); Gruber et al.(1991). Current Micro. 22: 15-20; Jarvis et al. (1991) Current Genet.19: 317-322 (Chlorella); for additional promoters see also Table 1 fromU.S. Pat. No. 6,027,900).

The promoter used to express an exogenous gene can be the promoternaturally linked to that gene or can be a heterologous gene. Somepromoters are active in more than one species of microalgae. Otherpromoters are species-specific. Preferred promoters include promoterssuch as RBCS2 from Chlamydomonas reinhardtii and viral promoters, suchas cauliflower mosaic virus (CMV) and chlorella virus, which have beenshown to be active in multiple species of microalgae (see for examplePlant Cell Rep. 2005 March; 23(10-11):727-35; J. Microbiol. 2005 August;43(4):361-5; Mar Biotechnol (NY). 2002 January; 4(1):63-73). In otherembodiments, the Botryococcus malate dehydrogenase promoter, such anucleic acid comprising any part of SEQ ID NO:1, or the Chlamydomonasreinhardtii RBCS2 promoter (SEQ ID NO:2) can be used. Optionally, atleast 10, 20, 30, 40, 50, or 60 nucleotides or more of these sequencescontaining a promoter are used.

Promoters, cDNAs, and 3'UTRs, as well as other elements of the vectors,can be generated through cloning techniques using fragments isolatedfrom native sources (see for example Molecular Cloning: A LaboratoryManual, Sambrook et al. (3d edition, 2001, Cold Spring Harbor Press; andU.S. Pat. No. 4,683,202). Alternatively, elements can be generatedsynthetically using known methods (see for example Gene. 1995 Oct. 16;164(1):49-53).

Examples of genes encoding carbohydrate transporters to facilitate theuptake of exogenously provided carbohydrates include SEQ ID NOs: 3, 4,5, 6 and 7 and allelic or species variants thereof. Other variantshaving a nucleic acid sequence encodes a protein with at least about 60%amino acid sequence identity with a protein with a sequence representedby one of SEQ ID NOs: 3-7. Optionally, the nucleic acid sequence encodesa protein with at least about 85%, at least about 90%, at least about95%, or at least about 98%, or higher, amino acid identity with asequence of these SEQ ID NOs: 3-7.

Additional examples include a Chlorella hexose transporter (such asGenbank Q39525), a yeast hxt2 transporter (such as Genbank P23585), ahuman GLUT1 (such as Genbank AAA52571), a Nicotiana tabacum glucosetransporter (such as Genbank CAA47324), and a Vicia faba glucosetransport protein (such as Genbank CAB07812).

Alternatively or additionally to transformation, cells can bemutagenized and then selected for the ability to grow in the absence oflight energy but in the presence of a fixed carbon source. Examples ofmutagenesis include contact or propagation in the presence of a mutagen,such as ultraviolet light, nitrosoguanidine, and/or ethane methylsulfonate (EMS).

As one example, a method of the disclosure comprises providing a nucleicacid encoding a carbohydrate transporter protein containing codonspreferred in Botryococcus braunii; transforming a Botryococcus brauniicell with the nucleic acid; and selecting for the ability to undergocell division in the absence of light and in the presence of acarbohydrate that is transported by the carbohydrate transporterprotein. In another example, a method comprises subjecting a microalgalcell to a mutagen; placing the cell in the presence of a fixed carbonmolecule; and selecting for the ability to undergo cell division in theabsence of light.

Cells can be transformed by, e.g., biolistics, electroporation, glassbead transformation and silicon carbide whisker transformation,including those referenced previously in this section.

V Engineering Cells to Increase Hydrocarbon Production

Some wild-type cells already have good growth characteristics but do notproduce high yields of microrganisms or do not produce the desired typeof hydrocarbons. Examples include Pyrobotrys, Phormidium, Agmenellum,Carteria, Lepocinclis, Pyrobotrys, Nitzschia, Lepocinclis, Anabaena,Euglena, Spirogyra, Chlorococcum, Tetraedron, Oscillatoria, Phagus, andChlorogonium, which have the desirable growth characteristic of growingin municipal sewage or wastewater. Such cells can be engineered to haveimproved hydrocarbon production characteristics. Desired characteristicsinclude optimizing hydrocarbon yield per unit volume and/or per unittime, carbon chain length (e.g., for gasoline production), reducing thenumber of double or triple bonds, optionally to zero, removing oreliminating rings and cyclic structures, increasing the hydrogen:carbonratio of a particular species of hydrocarbon or of a population ofdistinct hydrocarbons, and removing oxygen atoms such as in the case ofan aldehyde decarbonylase. The engineering involves transforming one ormore genes encoding hydrocarbon modification enzymes such as, forexample, a squalene synthase gene (see GenBank Accession numberAF205791), an aldehyde decarbonylase (see GenBank Accession numbersBAA11024 and CAA03710).

TABLE III Examples of Hydrocarbon Modification Enzymes A. amino acidsequences contained, referenced, or encoded by nucleic acid sequencescontained or referenced in any of U.S. Pat. Nos.: 6,610,527 6,451,5766,429,014 6,342,380 6,265,639 6,194,185 6,114,160 6,083,731 6,043,0725,994,114 5,891,697 5,871,988 6,265,639 B. amino acid seqeunces ofGenBank accession numbers: AAO18435 ZP_00513891 Q38710 AAK60613 AAK60610AAK60611 NP_113747 CAB75874 AAK60612 AAF20201 BAA11024 AF205791 CAA03710

Each of the amino acid sequences contained or encoded by nucleic acidsequences contained in the U.S. Patent identified in Table IIIA ishereby incorporated by reference herein. Each of the amino acidsequences identified by the GenBank accession numbers in Table IIIB ishereby incorporated by reference herein.

Such genes can be obtained from cells already known to have goodhydrocarbon production such as Botryococcus braunii. Genes already knownto have a role in hydrocarbon production, e.g., a gene encoding anenzyme that saturates double bonds, can be transformed individually intorecipient cells. However, to practice the invention it is not necessaryto make a priori assumptions as to which genes are required. A libraryof DNA containing different genes, such as cDNAs from a good hydrocarbonproduction organism, can be transformed into recipient cells. The cDNAis preferably in operable linkage with a promoter active in microalgae.Examples of organisms that produce useful hydrocarbons are microalgaesuch as Botryococcus braunii, Dunaliella and Nannochloropsis, cells fromany Pinaceae organism and subclasses thereof, such as Abies, Picea,Pinus (such as Pinus jeffreyi), Stobus, and Tsuga, and otherhydrocarbon-producing organisms such as Pisum sativum. Differentrecipient microalgae cells transformed by a library receive differentgenes from the library. For example, a population of Botryococcus cellstransformed with a cDNA library from Pinus jeffreyi, which producesn-heptane, a high-energy alkane (C₇H₁₆), can be screened for a phenotypesuch as increased total hydrocarbon production, increased energy contentof a crude oil preparation of a given volume compared to a similarlyprepared crude hydrocarbon preparation from cells not transformed withthe cDNA library, and/or direct production of n-heptane. Transformantshaving improved hydrocarbon production are identified though screeningmethods known in the art, such as, for example, HPLC, gaschromatography, and mass spectrometry methods of hydrocarbon analysis(for examples of such analysis, see Biomass and Bioenergy Vol. 6. No. 4.pp. 269-274 (1994); Experientia 38; 47-49 (1982); and Phytochemistry 65(2004) 3159-3165). These transformants are then subjected to furthertransformation with the original library and/or optionally interbred togenerate a further round of organisms having improved hydrocarbonproduction. In a preferred embodiment, Botryococcus braunii cells thatare capable of heterotrophic growth and contain a functionalcarbohydrate transporter are transformed with a single exogenous gene ora cDNA library from a hydrocarbon-producing organism. General proceduresfor evolving whole organisms to acquire a desired property are describedin e.g., U.S. Pat. No. 6,716,631. Such methods entail, e.g., introducinga library of DNA fragments into a plurality of cells, whereby at leastone of the fragments undergoes recombination with a segment in thegenome or an episome of the cells to produce modified cells. Themodified cells are then screened for modified cells that have evolvedtoward acquisition of the desired function. Vectors and methods fortransformation are analogous to those discussed in connection withtrophic conversion.

Some microalgae produce significant quantities of polysaccharides inaddition to hydrocarbons. Because polysaccharide biosynthesis can use asignificant proportion of the total metabolic energy available to cells,mutagenesis of hydrocarbon-producing cells followed by screening forreduced or eliminated polysaccharide production generates novel strainsthat are capable of producing higher yields of hydrocarbons. Forexample, Botryococcus cells are known to produce extracellularpolysaccharide.

The phenol: sulfuric acid assay detects carbohydrates (see Hellebust,Handbook of Phycological Methods, Cambridge University Press, 1978; andCuesta G., et al., J Microbiol Methods. 2003 January; 52(1):69-73). The1,6 dimethylmethylene blue assay detects anionic polysaccharides. (seefor example Braz J Med Biol Res. 1999 May; 32(5):545-50; Clin Chem. 1986November; 32(11):2073-6).

Polysaccharides can also be analyzed through methods such as HPLC, sizeexclusion chromatography, and anion exchange chromatography (see forexample Prosky L, Asp N, Schweizer T F, DeVries J W & Furda I (1988)Determination of insoluble, soluble and total dietary fiber in food andfood products: Interlaboratory study. Journal of the Association ofOfficial Analytical Chemists 71, 1017±1023; Int J Biol Macromol. 2003November; 33(1-3):9-18). Polysaccharides can also be detected using gelelectrophoresis (see for example Anal Biochem. 2003 Oct. 15;321(2):174-82; Anal Biochem. 2002 Jan. 1; 300(1):53-68).

VI Culturing Microorganisms

Microrganisms are cultured both for purposes of conducting geneticmanipulations and for subsequent production of hydrocarbons. The formertype of culture is conducted on a small scale and initially, at leastunder conditions in which the starting microorganism can grow. Forexample, if the starting microorganism is a photoautotroph the initialculture can be conducted in the presence of light. The cultureconditions can be changed as the microorganism is evolved or engineeredto grow independently of light. Culture for purposes of hydrocarbonproduction is usually conducted on a large scale. Preferably a fixedcarbon source is present. The culture can also be exposed to light someor all of the time. In certain embodiments, a photoautotroph, such asBotryococcus braunii can be grown on a fixed carbon source, in theabsence of light.

Microalgae can be cultured in liquid media. The culture can be containedwithin a fermentor or bioreactor. In particular embodiments, where lightis not needed or not desired for growth, the fermentor or bioreactordoes not allow light to enter. Alternatively, microalgae can also becultured in a photofermentor or photobioreactor that contains a fixedcarbon source and allow light to strike the cells. In certainembodiments, exposure of microalgae cells to light, even in the presenceof a fixed carbon source that the cells transport and utilize (ie:mixotrophic growth), nonetheless accelerates growth compared toculturing cells in the dark. This is not necessarily true forBotryococcus braunii, which, in specific embodiments, is preferablygrown in the dark when a fixed carbon source is present. Culturecondition parameters can be manipulated to optimize total hydrocarbonproduction, the combination of hydrocarbon species produced, and/orproduction of a hydrocarbon species. In some instances it is preferableto culture cells in the dark, such as, for example, when using extremelylarge (40,000 liter and higher) fermentors or bioreactors that do notallow light to strike the culture or when culturing Botryococcus brauniiin the presence of a fixed carbon source.

Microalgal culture media typically contains components such as a fixednitrogen source, trace elements, optionally a buffer for pH maintenance,and phosphate. Other components can include a fixed carbon source suchas acetate or glucose, and salts such as sodium chloride, particularlyfor seawater microalgae. Examples of trace elements include zinc, boron,cobalt, copper, manganese, and molybdenum in, for example, therespective forms of ZnCl₂, H₃BO₃, COCl₂.6H₂O, CuCl₂.2H₂O, MuCl₂.4H₂O and(NH₄)₆MO₇O₂₄.4H₂O.

For organisms able to grow on a fixed carbon source, the fixed carbonsource can be, for example, a carbohydrate, such as, but not limited to,glucose, fructose, sucrose, galactose, xylose, mannose, or rhamnose;N-acetylglucosamine; glycerol; floridoside; and/or glucuronic acid. Theone or more carbon source(s) can be supplied at a concentration of atleast about 50 μM, at least about 100 μM, at least about 500 μM, atleast about 5 mM, at least about 50 mM, at least about 500 mM, or at aconcentration within any range having any of these values as endpoints,of one or more exogenously provided fixed carbon source(s). Expressed asa percentage of the culture medium, the one or more carbon sources canbe supplied at a concentration of about 0.1%, about 0.2%, about 0.3%,about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%,about 1.0%, about 1.2%, about 1.5%, about 1.7%, about 2%, about 3%,about 4%, about 5%, or at a concentration within any range having any ofthese values as endpoints. The carbon source can be provided to themedium at these concentrations, without further addition of carbonsource. Alternatively, the concentration of the carbon source(s) can becontrolled during culture to be within a range having any of thesevalues as endpoints. Some microalgae species, e.g., Botryococcusbraunii, can grow by utilizing a fixed carbon source such as glucose inthe absence of light. Such growth is known as heterotrophic growth.

Some microorganisms naturally grow on or can be engineered to grow on afixed carbon source that is a heterogeneous source of compounds such asmunicipal waste, secondarily treated sewage, wastewater, and othersources of fixed carbon and other nutrients such as sulfates,phosphates, and nitrates. Microorganisms that are grown in media thatcomprises sewage, such as secondarily treated sewage are particularlyuseful. The sewage component serves as a nutrient source in theproduction of hydrocarbons, and the culture provides an inexpensivesource of hydrocarbons.

Other culture parameters can also be manipulated, such as the pH of theculture media, the identity and concentration of trace elements andother media constituents.

Microalgae can be grown in the presence of light. The number of photonsstriking a culture of microalgae cells can be manipulated, as well asother parameters such as the wavelength spectrum and ratio of dark:lighthours per day. Microalgae can also be cultured in natural light, as wellas simultaneous and/or alternating combinations of natural light andartificial light. For example, microalgae of the genus Chlorella can becultured under natural light during daylight hours and under artificiallight during night hours.

The gas content of a fermentor, bioreactor, photofermentor, orphotobioreactor to grow microorganisms like microalgae can bemanipulated. Part of the volume of a photobioreactor can contain gasrather than liquid. Gas inlets can be used to pump gases into thephotobioreactor. Any gas can be pumped into a photobioreactor, includingair, air/CO₂ mixtures, noble gases such as argon and others. The rate ofentry of gas into a fermentor, bioreactor, photofermentor, orphotobioreactor can also be manipulated. Increasing gas flow into afermentor, bioreactor, photofermentor, or photobioreactor increases theturbidity of a culture of microalgae. Placement of ports conveying gasesinto a fermentor, bioreactor, photofermentor, or photobioreactor canalso affect the turbidity of a culture at a given gas flow rate. Air/CO₂mixtures can be modulated to generate optimal amounts of CO₂ for maximalgrowth by a particular organism. Microalgae grow significantly faster inthe light under, for example, 3% CO₂/97% air than in 100% air. 3%CO₂/97% air is approximately 100-fold more CO₂ than found in air. Forexample, air:CO₂ mixtures of about 99.75% air:0.25% CO₂, about 99.5%air:0.5% CO₂, about 99.0% air:1.00% CO₂, about 98.0% air:2.0% CO₂, about97.0% air:3.0% CO₂, about 96.0% air:4.0% CO₂, and about 95.00% air:5.0%CO₂ can be infused into a fermentor, bioreactor, photofermentor, orphotobioreactor.

Microalgae cultures can also be subjected to mixing using devices suchas spinning blades and impellers, rocking of a culture, stir bars,infusion of pressurized gas, and other instruments. Optionally, afermentor, bioreactor, photofermentor, or photobioreactor apparatus ofthe invention comprises one or more of these devices such that thedevice(s) may be permanently attached to the apparatus or may beseparate initially but are later joined to form a complete apparatus.

The invention includes a fermentor, bioreactor, photofermentor, orphotobioreactor comprising a culture medium containing a fixed carbonsource and a hydrocarbon-producing microalgae as described herein.Optionally, the fixed carbon source is a carbohydrate, such as amonosaccharide or disaccharide as a non-limiting example. Non-limitingexamples include glucose, mannose, galactose, fructose, xylose,arabinose, sucrose, or other carbohydrates in Table II herein. In someembodiments, the microalgae is capable of metabolizing the carbohydrateor monosaccharide as a carbon source. Non-limiting examples includerecombinantly modified microalgae that are able to utilize thecarbohydrate or monosaccharide as a fixed carbon source. In otherembodiments, the microalgae is part of a combination of the inventioncomprising a first microalgae and a second microbe. The mixture ofmedium, carbohydrate, and microalgae (and optional second microbe) maybe in a vessel, or first location, of the fermentor, bioreactor,photofermentor, or photobioreactor.

A fermentor, bioreactor, photofermentor, or photobioreactor may be partof a system of the invention. The system may comprise a number ofvessels. A first vessel may be one in which a polysaccharide ishydrolyzed by an enzyme into monosaccharides. Alternatively,monosaccharides or a monosaccharide:oligosaccharide mixture such asdepolymerized cellulose can be provided directly into the system. Asecond vessel may be one in which the monosaccharides ormonosaccharide:oligosaccharide mixture are incubated with ahydrocarbon-producing microalgae capable of using the monosaccharides ormonosaccharide:oligosaccharide mixture as a fixed carbon source toproduce microalgal biomass. A third vessel may be one in which distinctspecies of hydrocarbons that have been extracted from the microalgalbiomass are separated or fractionated from each other. In someembodiments, the distinct species of hydrocarbons are separated orfractionated based upon the boiling temperatures of each species, suchas a distillation column, also known as a fractional distillationcolumn. For example, oil refineries use distillation columns tofractionate crude oil into different products. Distillation columns usedin oil refineries are typically large, vertical cylindrical columns withdiameters ranging from about 65 centimeters to 6 meters and heightsranging from about 6 meters to 60 meters or more. The distillationcolumns have liquid outlets at intervals up the column which allow forthe withdrawal of different fractions or products having differentboiling points or boiling ranges. The “lightest” products (those withthe lowest boiling point) exit from the top of the columns and the“heaviest” products (those with the highest boiling point) exit from thebottom of the column. Fractional distillation is used in oil refineriesto separate crude oil into useful substances (or fractions) havingdifferent hydrocarbons of different boiling points.

Methods for the growth and propagation of Botryococcus braunii are known(see for example Largeau et al., Phytochemistry, 1980, 19:1043-1051 andMetzger et al. Phytochemistry, 1985, 24(10):2305-2312). The inventionalso provides novel growth conditions for Botryococcus. For example,Botryococcus braunii can be grown in the dark in the presence of a fixedcarbon source. Alternatively or additionally, this species can be grownunder conditions comprising an increased amount of cobalt, which can bean essential factor in the synthesis of long chain hydrocarbons. Anincreased amount of cobalt above about 5 nM, above about 10 nM, aboveabout 100 nM, above about 1 μM, above about 10 μM, above about 100 μM,above about 1 mM, above about 10 mM, and above about 100 mM elementalcobalt can be used. Cobalt can be provided to cells in the form of, forexample, CoCl₂.6H₂O.

For hydrocarbon production, cells, including recombinant cells of theinvention described herein, are preferably cultured or fermented inlarge quantities. The culturing may be in large liquid volumes, such asin suspension cultures as an example. Other examples include startingwith a small culture of cells which expand into a large biomass incombination with cell growth and propagation as well as hydrocarbonproduction. Bioreactors or steel fermentors can be used to accommodatelarge culture volumes. A fermentor similar those used in the productionof beer and/or wine is suitable, as are extremely large fermentors usedin the commercial production of ethanol.

Appropriate nutrient sources for culture in a fermentor are provided.These include raw materials such as one or more of the following: afixed carbon source such as glucose, corn starch, cellulose,depolymerized cellulose as described herein (comprising a mixture ofglucose and xylose), sucrose, sugar cane, sugar beet, lactose, milkwhey, or molasses; a fat source, such as fats or vegetable oils; anitrogen source, such as protein, soybean meal, cornsteep liquor,hydrolyzed casein, urea, ammonia (pure or in salt form), nitrate ornitrate salt, or molecular nitrogen; and a phosphorus source, such asphosphate salts. Additionally, a fermenter allows for the control ofculture conditions such as temperature, pH, oxygen tension, and carbondioxide levels. Optionally, gaseous components, like oxygen or nitrogen,can be bubbled through a liquid culture. In certain embodiments,Botryococcus braunii is cultured in the dark with a fixed carbon sourceand a complex nitrogen source, such as hydrolyzed casein, and/or urea.

Thus the invention includes a method for producing hydrocarbons via afermentor or bioreactor with one or more of the above features, such asgrowth in the absence of light, the inclusion of cobalt in the cultureconditions; inoculation with a hydrocarbon-producing microalgae, such asBotryococcus braunii or one or more recombinant cells of the inventiondescribed herein; and use of appropriate nutrient sources, including afixed carbon source, a nitrogen source, and a phosphorus source. Ofcourse a method for producing hydrocarbons may comprise a combination oftwo or more, or all of the above, features.

In some embodiments, the method comprises a) providing culture mediathat comprised a carbohydrate or one or more species of monosaccharidesas a fixed carbon source, in a fermenter; b) inoculating the fermenterwith a hydrocarbon-producing microalgae capable of metabolizing thecarbohydrate or monosaccharide(s) as a carbon source; c) culturing themicroalgae for a period of time to generate microalgal biomass; d)separating or extracting or isolating hydrocarbons from the microalgalbiomass; and e) refining the separated, extracted, or isolatedhydrocarbons. In an alternative embodiment, the method comprises a)combining, in a fermentor, a carbohydrate or one or more species ofmonosaccharides in a culture medium with a hydrocarbon-producingmicroalgae capable of metabolizing the carbohydrate or monosaccharide(s)as a carbon source to form a mixture; b) culturing the microalgae insaid mixture to produce hydrocarbon containing microalgal biomass; c)separating or isolating hydrocarbons from said biomass; and d) refiningthe separated or isolated hydrocarbons.

Non-limiting examples of a monosaccharide for use in a disclosed methodinclude glucose, xylose, and arabinose. A disaccharide that can be usedis sucrose. In other embodiments, the carbohydrate is selected fromTable II herein. Non-limiting examples of a fermenter include aphotobioreactor or a fermenter that allows culturing of the microalgaewithout light exposure thereto. The microalgae may thus be culturedwithout light that strikes them or in the absence of light.

In many embodiments, the microalgae is selected from Table I herein. Insome methods, the microalgae is Botryococcus braunii. In otherembodiments, the microalgae has been transformed with an exogenous geneencoding a carbohydrate transporter as described herein.

In further embodiments, the method may further comprise the hydrolysisof a polysaccharide to produce a carbohydrate or monosaccharide for theculture media. The hydrolysis may be by any methodology known to theskilled person, including, but not limited to, enzyme catalyzedhydrolysis. In some embodiments, the hydrolysis is mediated by one ormore enzymes, such as, but not limited to, the 74polysaccharide-degrading enzymes from Aspergillus nidulans, Aspergillusfumigatus, and Neurospora crassa as described by Bauer et al.(“Development and application of a suite of polysaccharide-degradingenzymes for analyzing plant cell walls.” Proc Natl Acad Sci USA. 2006,103(30):11417-22. Epub 2006 Jul. 14). Non-limiting examples of apolysaccharide for use in the method include corn starch and cellulose.In some cases, the method comprises the use of corn starch or celluloseas the polysaccharide and enzymes which degrade it to produce glucose.

The separating, extracting, or isolating of hydrocarbons from thebiomass may by via any methodology known to the skilled person.Non-limiting examples include the harvesting methodologies describedbelow. In some embodiments, the methodology comprises hexane extraction,pressing the biomass, or by in vivo extraction (see for example,European Patent Application EP20030721175 entitled “Process forcontinuous production and extraction of carotenoids from naturalsources.” and discussion of in vivo extraction from living cells in thesection below). The separated, extracted, or isolated hydrocarbons maybe refined by any methodology known to the skilled person. Non-limitingexamples of refining include cracking the hydrocarbons, as describedherein, and the separating of different hydrocarbon species by use of afractional distillation column.

A fermentor or bioreactor can be used to allow cells to undergo thevarious phases of their growth cycle. As an example, an inoculum ofhydrocarbon-producing cells can be introduced into a medium followed bya lag period (lag phase) before the cells begin growth. Following thelag period, the growth rate increases steadily and enters the log, orexponential, phase. The exponential phase is in turn followed by aslowing of growth due to decreases in nutrients and/or increases intoxic substances. After this slowing, growth stops, and the cells entera stationary phase or steady state. The amount of biomass in stationaryphase is generally constant unless there is cell lysis.

Hydrocarbon production by cells disclosed herein occur mainly during thelog phase and sometimes thereafter, including the stationary phasewherein nutrients are supplied, or still available, to allow thecontinuation of hydrocarbon production in the absence of cell growth.

Mixotrophic growth is the use of both light and fixed carbon source(s)as energy sources for cells to grow and produce hydrocarbons.Mixotrophic growth can be conducted in a photofermentor orphotobioreactor. Microalgae can be grown and maintained in closedphotobioreactors made of different types of transparent orsemitransparent material. Such material can include Plexiglas®enclosures, glass enclosures, bags made from substances such aspolyethylene, transparent or semitransparent pipes, and other materials.Microalgae can be grown and maintained in open photofermentors orphotobioreactors such as raceway ponds, settling ponds, and othernon-enclosed containers.

Fermentors, bioreactors, photofermentors, or photobioreactors can haveports allowing entry of gases, solids, semisolids and liquids into thechamber containing the microalgae. Ports are usually attached to tubingor other means of conveying substances. Gas ports, for example, conveygases into the culture. Pumping gases into a fermentor, bioreactor,photofermentor, or photobioreactor can serve to both feed cells CO₂ andother gases and to aerate the culture and therefore generate turbidity.The amount of turbidity of a culture varies as the number and positionof gas ports is altered. For example, gas ports can be placed along thebottom of a cylindrical polyethylene bag. Microalgae grow faster whenCO₂ is added to air and bubbled into a photobioreactor. For example, a5% CO₂:95% air mixture is infused into a fermentor, bioreactor,photofermentor, or photobioreactor containing Botryococcus cells (seefor example J Agric Food Chem. 2006 Jun. 28; 54(13):4593-9; J BiosciBioeng. 1999; 87(6):811-5; and J Nat. Prod. 2003 June; 66(6):772-8).

Photobioreactors or photofermentors can be exposed to one or more lightsources to provide microalgae with light as an energy source via lightdirected to a surface of the photobioreactor or photofermentor.Preferably the light source provides an intensity that is sufficient forthe cells to grow, but not so intense as to cause oxidative damage orcause a photoinhibitive response. In some instances a light source has awavelength range that mimics or approximately mimics the range of thesun. In other instances a different wavelength range utilized by themicroalgae is used. Photobioreactors or photofermentors can be placedoutdoors or in a greenhouse or other facility that allows sunlight tostrike the surface. Preferred photon intensities for species of thegenus Botryococcus are between 25 and 500 μE m⁻² s⁻¹ (see for examplePhotosynth Res. 2005 June; 84(1-3):21-7).

Fermentors, bioreactors, photofermentors, or photobioreactors preferablyhave one or more ports that allow media entry. It is not necessary thatonly one substance enter or leave a port. For example, a port can beused to flow culture media into the fermentor, bioreactor,photofermentor, or photobioreactor and then later can be used forsampling, gas entry, gas exit, or other purposes. In some instances afermentor, bioreactor, photofermentor, or photobioreactor is filled withculture media at the beginning of a culture and no more growth media isinfused after the culture is inoculated. In other words, the microalgalbiomass is cultured in an aqueous medium for a period of time duringwhich the microalgae reproduce and increase in number; howeverquantities of aqueous culture medium are not flowed through thefermentor, bioreactor, photofermentor, or photobioreactor throughout thetime period. Thus in some embodiments, aqueous culture medium is notflowed through the fermentor, bioreactor, photofermentor, orphotobioreactor after inoculation.

In other instances culture media can be flowed though the fermentor,bioreactor, photofermentor, or photobioreactor throughout the timeperiod during which the microalgae reproduce and increase in number. Insome embodiments media is infused into the fermentor, bioreactor,photofermentor, or photobioreactor after inoculation but before thecells reach a desired density. In other words, a turbulent flow regimeof gas entry and media entry is not maintained for reproduction ofmicroalgae until a desired increase in number of said microalgae hasbeen achieved.

Fermentors, bioreactors, photofermentors, or photobioreactors preferablyhave one or more ports that allow gas entry. Gas can serve to bothprovide nutrients such as CO₂ as well as to provide turbulence in theculture media. Turbulence can be achieved by placing a gas entry portbelow the level of the aqueous culture media so that gas entering thefermentor, bioreactor, photofermentor, or photobioreactor bubbles to thesurface of the culture. One or more gas exit ports allow gas to escape,thereby preventing pressure buildup in the fermentor, bioreactor,photofermentor, or photobioreactor. Preferably a gas exit port leads toa “one-way” valve that prevents contaminating microorganisms to enterthe fermentor, bioreactor, photofermentor, or photobioreactor. In someinstances cells are cultured in a fermentor, bioreactor, photofermentor,or photobioreactor for a period of time during which the microalgaereproduce and increase in number; however a turbulent flow regime withturbulent eddies predominantly throughout the culture media caused bygas entry is not maintained for all of the period of time. In otherinstances a turbulent flow regime with turbulent eddies predominantlythroughout the culture media caused by gas entry can be maintained forall of the period of time during which the microalgae reproduce andincrease in number. In some instances a predetermined range of ratiosbetween the scale of the fermentor, bioreactor, photofermentor, orphotobioreactor and the scale of eddies is not maintained for the periodof time during which the microalgae reproduce and increase in number. Inother instances such a range can be maintained.

Fermentors, bioreactors, photofermentors, or photobioreactors preferablyhave at least one port that can be used for sampling the culture.Preferably a sampling port can be used repeatedly without compromisingthe axenic nature of the culture. A sampling port can be configured witha valve or other device that allows the flow of sample to be stopped andstarted. Alternatively a sampling port can allow continuous sampling.Fermentors, bioreactors, photofermentors, or photobioreactors preferablyhave at least one port that allows inoculation of a culture. Such a portcan also be used for other purposes such as media or gas entry.

VII Harvesting

Hydrocarbons produced by cells of the invention can be harvested, orotherwise collected, by any convenient means. For example, hydrocarbonssecreted from cells can be centrifuged to separate the hydrocarbons in ahydrophobic layer from contaminants in an aqueous layer and optionallyfrom any solid materials as a precipitate after centrifugation.Extracellular hydrocarbons can also be separated by tangential flowfiltration. Preferred organisms for culturing in fermentors,bioreactors, photofermentors, or photobioreactors to producehydrocarbons include those disclosed herein. Material containing cell orcell fractions can be treated with proteases to degrade contaminatingproteins before or after centrifugation. In some instances thecontaminating proteins are associated, possibly covalently, tohydrocarbons or hydrocarbon precursors which form hydrocarbons uponremoval of the protein. In other instances the hydrocarbon molecules arein a preparation that also contains proteins. Proteases can be added tohydrocarbon preparations containing proteins to degrade proteins (forexample, the protease from Streptomyces griseus can be used(SigmaAldrich catalog number P5147). After digestion, the hydrocarbonsare preferably purified from residual proteins, peptide fragments, andamino acids. This purification can be accomplished, for example, bymethods listed above such as centrifugation and filtration.

Hydrocarbons can also be isolated by whole cell extraction. The cellsare first disrupted and then intracellular and cell membrane/cellwall-associated hydrocarbons as well as extracellular hydrocarbons canbe collected from the whole cell mass, such as by use of centrifugationas described above.

Alternatively, cells can be homogenized to facilitate hydrocarboncollection. As a non-limiting example, a pressure disrupter can be usedto pump a cell-containing slurry through a restricted orifice valve.High pressure (up to 1500 bar) is applied, followed by an instantexpansion through an exiting nozzle. Cell disruption is accomplished bythree different mechanisms: impingement on the valve, high liquid shearin the orifice, and sudden pressure drop upon discharge, causing anexplosion of the cell. The method releases intracellular molecules.

Alternatively, a ball mill can be used. In a ball mill, cells areagitated in suspension with small abrasive particles. Cells breakbecause of shear forces, grinding between beads, and collisions withbeads. The beads disrupt the cells to release cellular contents. In someembodiments, a sample is cryogenically ball milled in a planetary ballmill (Retsch, PM100) at 10-80 grams per batch size. The powder is placedin a grinding bowl with eight to ten ¾-inch-diameter stainless steelballs. The sample is cooled repeatedly with liquid nitrogen. Thematerial was milled at 400-550 rpm for about 30 to about 60 min. Thefinal product was dried in a desiccator overnight.

Cells can also by lysed with high frequency sound. The sound can beproduced electronically and transported through a metallic tip to anappropriately concentrated cellular suspension. This sonication (orultrasonication) disrupts cellular integrity based on the creation ofcavities in cell suspension. Cells can also be disrupted by shearforces, such as with the use of blending (such as with a high speed orWaring blender as examples), the french press, or even centrifugation incase of weak cell walls, to disrupt cells.

Various methods are available for separating hydrocarbons from cellularlysates produced by the above methods. For example, hydrocarbons can beextracted with a hydrophobic solvent like hexane (see Frenz et al. 1989,Enzyme Microb. Technol., 11:717). Hydrocarbons can also be extractedusing liquefaction (see for example Sawayama et al. 1999, Biomass andBioenergy 17:33-39 and Inoue et al. 1993, Biomass Bioenergy6(4):269-274); oil liquefaction (see for example Minowa et al. 1995,Fuel 74(12):1735-1738); and supercritical CO₂ extraction (see forexample Mendes et al. 2003, Inorganica Chimica Acta 356:328-334).

Hexane solvent extraction can be used in isolation or it can be usedalong with the oil press/expeller method. After the oil has beenextracted using an expeller, the remaining pulp can be mixed with hexaneto extract the remaining oil content. The oil dissolves in thecyclohexane, and the pulp is filtered out from the solution. The oil andhexane are then separated by means of distillation.

Another method of oil extraction is the supercritical fluid/carbondioxide extraction method, in which carbon dioxide is liquefied underpressure and heated to the point that it has the properties of both aliquid and gas. This liquefied fluid then acts as the solvent inextracting the oil from the algal biomass.

Solventless extraction of hydrocarbons can also be used, such as themethods described in U.S. Pat. No. 6,750,048.

Extracellular hydrocarbons can also be extracted in vivo from livingmicroalgae cells which are then returned to a fermentor or bioreactor byexposure of the cells, in an otherwise sterile environment, to anon-toxic extraction solvent, followed by separation of the living cellsand the hydrophobic fraction of extraction solvent and hydrocarbons,wherein the separated living cells are then returned to a culturecontainer such as a stainless steel fermenter or bioreactor orphotobioreactor or photofermentor (see Biotechnol Bioeng. 2004 Dec. 5;88(5):593-600 and Biotechnol Bioeng. 2004 Mar. 5; 85(5):475-81). Such invivo extraction is also described in European patent applicationEP20030721175 20030507 entitled “PROCESS FOR CONTINUOUS PRODUCTION ANDEXTRACTION OF CAROTENOIDS FROM NATURAL SOURCES”.

Hydrocarbons can also be extracted from algal biomass by pressing ofmaterial. When algae is dried it retains its oil content, which then canbe pressed out with an oil press. For example, commercial manufacturersof vegetable oil use a combination of mechanical pressing and chemicalsolvents in extracting oil. For representative oil presses, see IBGMonforts Oekotec GmbH & Co., Germany. Also see for example U.S. Pat.Nos. 5,186,722; 5,939,571; and 5,077,071.

VIII Modification of Hydrocarbons Produced by Cells

Hydrocarbons produced by cells as described herein can be modified bythe use of one or more enzymes. When the hydrocarbons are in theextracellular environment of the cells, one or more enzymes can be addedto that environment under conditions in which the enzyme modifies thehydrocarbon or completes its synthesis from a hydrocarbon precursor.Alternatively, the hydrocarbons can be partially, or completely,isolated from the cellular material before addition of one or moreenzymes. Such enzymes are exogenously added, and their enzymaticactivity occurs outside the cell or in vitro.

Suitable examples of enzymes for use in modifying hydrocarbons includethose which saturate carbon-carbon double, or triple, bonds inhydrocarbon molecules; and enzymes listed in Table III as well asallelic and species variants thereof. In further embodiments, the enzymeis squalene synthase, as GenBank accession number AAF20201, and speciesand allelic variants thereof, or other variants exhibiting at least 70%with AAF202201 and have squalene synthase activity. Alternatively, theenzyme can be a terpene synthase, such as, but not limited to, apolypeptide that has at least 70% amino acid identity with the sequencefound as GenBank accession numbers AAO18435, ZP_(—)00513891, and Q38710,or allelic or species variants of any of these, and exhibits terpenesynthase activity. Alternatively, the enzyme can be an aldehydedecarbonylase such as GenBank Accession numbers BAA11024 and CAA03710),or a polypeptide that has at least 70% amino acid identity with one ofGenBank Accession numbers BAA11024 or CAA03710 that exhibits aldehydedecarbonylase activity. In additional embodiments, the enzymaticactivity is present in a sequence that has at least about 70%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or at least about 99% identity with one of the above describedsequences, all of which are hereby incorporated by reference as if fullyset forth.

A substrate and/or cofactor of an enzyme can also be added for use incombination with the enzyme. Examples of a substrate or cofactor includeNADPH, NADH, ATP, or cobalt or cobalt ion. Optionally, the substrateand/or cofactor can be produced in situ by an added cell free extract.Optionally, the substrate and/or cofactor is produced by an addedproduct of co-cultured cells.

IX Additional Processing/Extraction

Hydrocarbons produced by cells in vivo, or enzymatically modified invitro, as described herein can be optionally further processed byconventional means. The processing can include “cracking” to reduce thesize, and thus increase the hydrogen:carbon ratio, of hydrocarbonmolecules. Catalytic and thermal cracking methods are routinely used inhydrocarbon processing. Catalytic methods involve the use of a catalyst,such as a solid acid catalyst. The catalyst can be silica-alumina or azeolite, which result in the heterolytic, or asymmetric, breakage of acarbon-carbon bond to result in a carbocation and a hydride anion. Thesereactive intermediates then undergo either rearrangement or hydridetransfer with another hydrocarbon. The reactions can thus regenerate theintermediates to result in a self-propagating chain mechanism.Hydrocarbons can also be processed to reduce, optionally to zero, thenumber of carbon-carbon double, or triple, bonds therein. Hydrocarbonscan also be processed to remove or eliminate a ring or cyclic structuretherein. Hydrocarbons can also be processed to increase thehydrogen:carbon ratio. This can include the addition of hydrogen(“hydrogenation”) and/or the “cracking” of hydrocarbons into smallerhydrocarbons.

Thermal methods involve the use of elevated temperature and pressure toreduce hydrocarbon size. An elevated temperature of about 800° C. andpressure of about 700 kPa can be used. These conditions generate“light,” a term that is sometimes used to refer to hydrogen-richhydrocarbon molecules (as distinguished from photon flux), while alsogenerating, by condensation, heavier hydrocarbon molecules which arerelatively depleted of hydrogen. The methodology provides homolytic, orsymmetrical, breakage and produces alkenes, which may be optionallyenzymatically saturated as described above.

Catalytic and thermal methods are standard in plants for hydrocarbonprocessing and oil refining. Thus hydrocarbons produced by cells asdescribed herein can be collected and processed or refined viaconventional means. See Hillen et al. (Biotechnology and Bioengineering,Vol. XXIV:193-205 (1982)) for a report on hydrocracking of B. brauniiproduced hydrocarbons. In alternative embodiments, the fraction istreated with another catalyst, such as an organic compound, heat, and/oran inorganic compound.

X Hydrocarbon Compositions

In certain embodiments, practice of the above methods results inhydrocarbon compositions different in type and/or quantity than thoseproduced by conventional methods. As discussed above, such compositionscan be provided purified in whole or in part from one or more componentsnormally found with the hydrocarbons. Examples include compositions ofmaterials from cell culture, which may include cells, cell fragments,intracellular components, and culture media components. Optionallycomponents of hydrocarbon compositions include botryococcene, squalene,and/or farnesyl diphosphate. Final products can include any of thefractions conventionally distilled from crude oil as discussed above. Asan example, co-expression of a glucose transporter and an aldehydedecarbonylase in B. braunii in the presence of exogenously providedglucose generates significantly more hydrocarbon molecules containingonly carbon and hydrogen per unit volume of culture per unit time thancan be produced by culturing wild-type B. braunii. The resulting, novelhydrocarbon compositions are an aspect of the invention. Expression ofan aldehyde decarbonylase by a promoter that is active constitutivelyallows for continuous catalytic transformation of numerous species ofaldehydes to alkanes at all phases of the cell cycle. Aldehydedecarbonylases catalyze the decarbonylation of aldehydes to form alkanesor alkenes and carbon monoxide. This reaction increases the overallenergy content of a hydrocarbon preparation containing aldehydes.

In vitro processing of hydrocarbons produced by microorganisms viaenzymes or other means is usually incomplete, giving rise to a mixedpopulation of hydrocarbons. Some of the hydrocarbons produced in such apopulation remain in the form produced in vivo by a microorganism. Otherhydrocarbons in the population initially produced in vivo have undergonefurther processing in vitro and differ from the hydrocarbons resultingsolely from in vivo processing.

All references cited herein, including patents, patent applications, andpublications, are hereby incorporated by reference in their entireties,whether previously specifically incorporated or not. The publicationsmentioned herein are cited for the purpose of describing and disclosingreagents, methodologies and concepts that may be used in connection withthe present invention. Nothing herein is to be construed as an admissionthat these references are prior art in relation to the inventionsdescribed herein.

Although this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

EXAMPLE 1

The methods here can be used to GE the bug with other valuable genessuch as: and name a few. Boiler plate out of the other application.

A Botryococcus braunii codon-optimized synthetic cDNA encoding theprotein of SEQ ID NO:3 is placed in operable linkage with a firstpromoter of SEQ ID NO:2. A cDNA identical to GenBank accession numberAF205791 (encoding squalene synthase) is cloned into the same plasmid inoperable linkage with a second promoter of SEQ ID NO:2.

The resulting nucleic acid vector is used to transform Botryococcusbraunii cells (strain UTEX-2441) via biolistic transformation asdescribed in Zaslayskaia et al. Science (2001) 292:2073-2075. Cells areplated on 4×mCHU agar media (see Phytochemistry, 1980, Vol. 19, pp.1043-1051) containing 0.5% glucose. Plates are stored in the dark.Colonies of trophically converted B. braunii are recovered from platesand streak purified on new plates to isolate single colonies.

A transgenic strain exhibiting heterotrophic growth is cultured in anErlenmeyer flask in the dark in 4×mCHU media containing 0.5% glucose.Also grown in parallel is a culture of wild type Botryococcus brauinii(UTEX 2441) in the presence of 125 μE/s/m² light. After the culturesreach plateau density they are harvested. Cell pellets are extractedwith hexane by the method of Biomass and Bioenergy, Vol. 6. No. 4., pp.269-274 (1994). Oil preparations are then analyzed by gas chromatographyas described in Phytochemistry 65 (2004) 3159-3165. The preparationsfrom the transgenic strain are a composition comprising hydrocarbonmolecules extracted from a plurality of transgenic microalgae cells,wherein each cell contains a first exogenous gene encoding acarbohydrate transporter that enables the cell to grow heterotrophicallyin the presence of a carbohydrate that is transported by the transporterand a second exogenous gene encoding a hydrocarbon modification enzyme;and the composition contains a novel hydrocarbon molecule not found innon-transgenic microalgae cells of the same species; and/or a greateramount of an endogenous hydrocarbon molecule per cell relative to otherhydrocarbons found in non-transgenic microalgae cells of the samespecies.

EXAMPLE 2 Heterotrophic Growth of Botryococcus braunii Background

Botryococcus braunii, a unicellular green alga, has been known to be anobligate autotroph. It was believed to require light energy to fix CO₂to grow or that genetic engineering of the microalgae with a sugar(e.g., hexose or pentose) transporter would be necessary to grow B.braunii in the dark. However, we found B. braunii can growheterotrophically with fixed carbon sources in the dark without the needfor genetic engineering. This is an important finding since it providesa new way to culture B. braunii and an opportunity to obtain highdensity cultures of B. braunii using fermentation equipment.

Strains

Three Botryococcus braunii strains (UTEX 572, UTEX 2441, and N-836).UTEX 572 and UTEX 2441 are available from The University of Texas atAustin, The Culture Collection of Algae (UTEX), 1 University StationA6700, Austin, Tex. 78712-0183 USA. N-836 is available from MICROBIALCULTURE COLLECTION, National Institute for Environmental Studies, 16-2Onogawa, Tsukuba, Ibaraki, 305-8506 JAPAN.

Media

Modified BG-11 (17.65 mM NaNO₃, 0.23 mM K2PO4, 0.3 mM MgSO₄.7H₂O, 0.25mM CaCl₂.2H₂O, 0.189 mM Na₂CO₃, 0.03 mM citric acid monohydrate, 0.023mM ferric ammonium citrate, 0.046 mM H₃BO₃, 9.15 μM MnCl₂.4H₂O, 0.77 μMZnSO₄.7H₂O, 0.32 μM CuSO₄.5H₂O, 0.21 μM CoCl₂.6H₂O, 1.62 μM NaMoO₄.2H₂O,2.69 μM Na₂EDTA, 9 mg/l Tricine, 1.99 μM Thiamine-HCl, 0.006 μMCyanocobalamine, 0.044 μM Calcium Pantothenate, 0.29 μM p-aminobenzoicacid, soil water). This was prepared by adding soil water and vitaminmix to basic BG11 for better growth support (recipe available fromAmerican Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va.20108 USA.

Mixotrophic Condition

In the past, growth with fixed carbon sources in the light has beenexplored. We have tested mannose, fructose, galactose, glycerol, acetateand glucose in BG11 media and cultured UTEX 2441 in light. In themixotrophic condition, fixed carbons other than glycerol showedinhibitory effects on growth. The experiment was repeated with UTEX 572and UTEX 2441 for glycerol. Whereas UTEX 572 showed a marginaldifference in growth with or without glycerol, UTEX 2441 showed 66%increased growth with glycerol. Dry cell weight (DCW) were measuredafter three weeks for the glycerol samples.

TABLE 4 Mixotrophic growth of B. braunii with glycerol A. UTEX 572 B.UTEX 2441 DCW mg/15 ml. DCW g/L A. 0.75% glycerol 4.1 0.273 0.75%glycerol 3.1 0.206 1% glycerol 4.0 0.267 1% glycerol 3.0 0.20 1.5%glycerol 3.6 0.24 1.5% glycerol 4.4 0.293 BG11 only 3.1 0.206 BG11 only2.7 0.18 BG11 only 3.3 0.22 B. 0.75% glycerol 4.1 0.273 0.75% glycerol4.3 0.286 1% glycerol 3.3 0.22 1% glycerol 4.8 0.32 1.5% glycerol 4.30.24 1.5% glycerol 4.7 0.286 BG11 only 1.7 0.113 BG11 only 1.4 0.093BG11 only 1.4 0.093Heterotrophic Growth of B. braunii

We tested heterotrophic growth of B. braunii with glucose. Thisexperiment was done in a 24 well plate with 1 ml of BG11 plus glucose inthe dark or light. Different concentrations of glucose (0.1, 0.5, 1%)were tested. Both strains clearly grew in the dark, showing best growthwith 1% glucose. However, no growth in either strains with glucose inthe light was observed. Dry cell weight (DCW) was used to measure cellgrowth after 4 weeks of growth (Table 5).

TABLE 5 DCW measurement of UTEX 572 (A) and UTEX 2441 (B) culture grownin the dark DCW mg/ml. DCW g/L A. 0.1% glucose 0.2 0.2 0.2% glucose 0.20.2 0.5% glucose 0.4 0.4 1.0% glucose ND^(†) no glucose −0.2 −0.2 B.0.1% glucose 0.3 0.3 0.2% glucose 0.3 0.3 0.5% glucose 0.1 0.1 1.0%glucose 0.7 0.7 no glucose 0 0 ^(†)culture used to inoculate 20 mLcultureImprovement of Heterotrophic Growth by Culture Passage

1 ml of UTEX 572 culture grown in 1% glucose in the dark from theprevious experiment was passaged three times in the dark with 1% glucosewhich yielded a higher density culture with better growth rate. Theheterotrophic culture was also expanded from 1 ml to 400 ml.

Optimization of Heterotrophic Growth of B. braunii

We tested complex nitrogen sources (urea and hydrolysate casein) onheterotrophic growth of B. braunii with glucose. The followingexperiment was done with UTEX 572, UTEX 2441, and N-836 in 20 ml of BG11plus:

1% glucose

1% glucose+2 g/L hydrolyzed casein

1% glucose+10 mM urea

After 4 weeks, UTEX 572 showed 76% increased growth with 10 mM urea overglucose only culture, and UTEX 2441 and N-836 showed increased growthwith 2 g/L hydrolysate casein to 340% and 61% respectively. Controlcultures of all three strains (BG11 only) showed no growth in the dark.The results are summarized below in Table 6.

TABLE 6 Effects of urea and hydrolysate casein on heterotrophic growthof B. braunii measured by DCW. A. UTEX 572, B. UTEX 2441, and C. N-836.DCW mg/3 m1. DCW g/L A. UTEX 572 1% glucose 0.6 0.20 1% glucose 1.0 0.331% glucose + 2 g/L hydrolyzed casein 0.5 0.167 1% glucose + 10 mM Urea1.4 0.467 BG11 control 0.2 0.066 B. UTEX 2441 1% glucose 0.2 0.067 1%glucose + 2 g/L hydrolyzed casein 0.9 0.30 1% glucose + 2 g/L hydrolyzedcasein 0.9 0.30 1% glucose + 10 mM Urea 0.3 0.10 1% glucose + 10 mM Urea0.2 0.067 BG11 control 0.2 0.067 C. N-836 1% glucose 0.8 0.267 1%glucose 0.8 0.267 1% glucose + 2 g/L hydrolyzed casein 1.0 0.33 1%glucose + 2 g/L hydrolyzed casein 1.6 0.53 1% glucose + 10 mM Urea 0.20.067 1% glucose + 10 mM Urea 0.9 0.30 BG11 control 0.3 0.10Test of Different Carbon Sources on Heterotrophic Growth of B. braunii.

We tested whether B. braunii can utilize different fixed carbon sources(mannose, glucose galactose, sodium acetate, fructose, glycerol andarabinose) for heterotrophic growth. All carbon sources were tested atthree different concentrations; 0.1%, 0.5% and 1%. Cultures were grownin 24 well plates for 4 weeks in the dark in 1 ml BG11 plus fixedcarbon. Control cultures were grown in BG-11 media under phototrophicconditions without a fixed carbon source. Growth was scored with +'s byvisual examination of the cultures (see Table 7). UTEX 572 grew best inglucose, however there was also growth on mannose, galactose andfructose. UTEX 2441 preferred mannose in the dark but there was somegrowth in galactose, fructose and glycerol. N-836 demonstratedheterotrophic growth with mannose, glucose, galactose and fructose.

TABLE 7 Effect of urea carbon source on heterotrophic growth of B.braunii. 0.1% 0.5% 1% 0.1% 0.5% 1% A. UTEX 572 mannose ++ ++ +++ ++ ++++ fructose glucose +++ ++++ +++++ + + + glycerol galactose ++ ++++++ + + + arabinose acetate + + + control control + + B. UTEX 2441mannose +++ ++++ +++++ ++ +++ +++ fructose glucose ++ ++ ++++ ++ +++ +++glycerol galactose ++ +++ ++ ++ ++ ++ arabinose acetate ++ ++ ++ controlcontrol ++ ++ C. N-836 mannose ++ ++ ++ + + ++ fructose glucose ++ +++++ + + + glycerol galactose ++ ++ ++ + + + arabinose acetate + + +control control + +

EXAMPLE 3 Media Optimization for Heterotrophic Growth of Botryococcusbraunii

Media Component Screening

Media components incorporated in soil bacteria media were tested inheterotrophic culture of Botryococcus braunii (strain UTEX 572). Foreach component tested, 5 ml of B11 media (as described in Example 2above) supplemented with 3% glucose (Fisher) were prepared in 6-wellplates. Additionally, the media was supplemented with 0.2% of one of thefollowing soil bacteria media components: dextrin (MP Biomedicals), maltextract (MP Biomedicals), traders yeast (Pharmamedia), corn meal, cornsteep powder (Marcor), whole dead yeast (Engevita), casein type M(Marcor), casein type B (Marcor), tomato paste, molasses, soyhydrolysate (MP Biomedicals), soy flour (Arrowhead Mills), corn starch(Sigma), and maltose (Fisher). Wells were inoculated and cultures weregrown at room temperature in the dark for 16 days. Visual observation ofthe plates revealed better growth with maltose, soy flour, malt extract,corn starch, corn meal and dextrin conditions as compared to glucoseonly control. All media were filter sterilized.

The cells from each condition were transferred into 150 mL T150 flaskscontaining 50 mL B11 media with 3% glucose and 0.2% of their respectivesupplemental component. Cultures were grown in the dark for one week at28° C. Cells were pelleted and fresh media (100 mL) were added to thecultures. The cultures were allowed to grow for another two weeks in thedark at 28° C. and then were collected and dry cell weights weremeasured. For most conditions, the addition of a 0.2% supplementalcomponent increased cell growth as compared to glucose control. Thecomponents that produced the greatest increase in growth were soy flour(2 fold increase), malt extract (2 fold increase), cornstarch (2.5 foldincrease) and cornmeal (3 fold increase).

High Surface to Volume Heterotrophic Cultivation of Botryococcusbraunii.

In order to develop a reproducible, scalable process for theheterotrophic cultivation of B. braunii, a low-volume, slow-mixingheterotrophic procedure was developed. Botryococcus braunii (UTEX 572)cultures were transferred from solid media plates into 6-well platesusing B-11 media supplemented with 3% glucose and was grown for one weekat room temperature in the dark. The cultures were then transferred into50 mL of fresh media in a 150 mL T-flask and agitated at 40 rpm on aplate shaker for one week at room temperature in the dark. The cultureswere then transferred into 100 mL of fresh media in a 500 mL T-flask andagitated at 30 rpm with a two-inch throw for one week at roomtemperature in the dark. Finally, the cultures were transferred into 350mL of fresh media in a Fernbach flask and agitated at 30 rpm with atwo-inch throw for one week at room temperature in the dark. In one setof cultures, the cells were transferred into 500 mL of fresh media in aFernbach flask (instead of 350 mL). Dry cell weights were collected forall cultures and compared. Cultures that were grown in a final volume of350 mL of media reached a DCW of about 9 grams per liter. Interestingly,the culture with a final volume of 500 mL of media had reduced growthand only reached a DCW of about 3 grams per liter. The results suggestthat the slow increase in volume of media is important to heterotrophicgrowth of B. braunii.

SEQUENCES SEQ ID NO 1: Botryococcus braunii malate dehydrogenase 5′ UTRaattggaaaccccgcgcaagaccgggttgtttggccgcctgaccggaaagggggggcctgtcccgaagggggtctatctcttgggggatgtcgggcgcggaaagtcgatgttgatggacctcttcttcgaccatgtcggggtcgaggccaagagccgcgtccatttcgccgagttcatgatggaggtgaatgaccgcatcgccaccgaacgcgccaagaagcgggcgaccgatcgcccccgtcgctgcagcccttgccgaggaagtccggctgctggcgttcgacgagatgatggtgacgaacagcccggacgcgatgatcctgtcgcggctgttcaccgcgctgatcgaggcgggggtgacgatcgtcaccacctccaaccggccgcccagggatctctataagaacgggctcaaccgcgagcatttcctgcccttcatcgcgctgatcgaggcgcggctggacgtgctggcgctgaacggcccgaccgactatcggcgcgaccggctggggcggctggacacgtggttggtgcccaatggccccaaggcgacgattaccttgtcggcggcgttcttccgcctgaccgactatccggtcgaggatgccgcgcatgtgccctctgaggacctgaaggtgggcgggcgcgtgctgaatgtccccaaggcgctgaagggcgtcgcggtcttctcgttcaagcggttgtgcggcgaagcgcggggggcggcggactatctggcggtcgcgcggggcttccacaccgtcatcctggtcggaatccccaagctgggggcggagaaccgcaacgaggcggggcgcttcgtccagctgatcgacgcgctctacgaacataaggtcaagctgctcgccgcagccgatgccagcccgccgaactctatgaaaccggcgacggccggttcgagtttgagcgcagatcagccggttggaagagatgcgctccgaggattatctggcccaaggccatggctcggaggggccttgatcaggccttaatgcacttcgcaaccattatcgtttaaaatcttaaactctgtggaataacggttccccgacgccgcaatacacgtacgtccactacggagtaggattggaSEQ ID NO 2: RBCS2 (Rubisco) Chlamydomonas reinhardtiicgcttagaagatttcgataaggcgccagaaggagcgcagccaaaccaggatgatgtttgatggggtatttgagcacttgcaacccttatccggaagccccctggcccacaaaggctaggcgccaatgcaagcagttcgcatgcagcccctggagcggtgccctcctgataaaccggccagggggcctatgttctttacttttttacaagagaagtcactcaacatcttaaacggt cttaagaagtctatccggSEQ ID NO 3: chlorella hexose transporter fromQ39525 Parachlorella kesslerimaggaivasggasrsseyqggltayvllvalvaacggmllgydngvtggvasmeqferkffpdvyekkqqivetspyctydnpklqlfvsslflagliscifsawitrnwgrkasmgiggiffiaagglvnafaqdiamlivgryllgfgvglgsqvvpqylsevapfshrgmlnigyqlfvtigiliaglvnygvrnwdngwrlslglaavpglilllgaivlpespnflvekgrtdqgrrileklrgtshveaefadivaaveiarpitmrqswrslftrrympqlltsfviqffqqftginaiifyvpvlfsslgsassaallntvvvgavnvgstmiavllsdkfgrrfllieggitcclamlaagitlgvefgqygtedlphpvsagvlavicifiagfawswgpmgwlipseiftletrpagtavavmgnflfsfvigqafvsmlcamkfgvflffagwlvimvlcaifllpetkgvpiervqalyarhwfwkkvmgpaaqellaedekrv aasqaimkeerisqtmkSEQ ID NO 4: glucose transporter [Arabidopsis thaliana]GlcGalFrc from CAA390 mpaggfvvgdgqkaypgkltpfvlftcvvaamgglifgydigisggvtsmpsflkrffpsvyrkqqedastnqycqydsptltmftsslylaalisslvastvtrkfgrrlsmlfggilfcagalingfakhvwmlivgrillgfgigfanqavplylsemapykyrgalnigfqlsitigilvaevinyffakikggwgwrlslggavvpaliitigslvlpdtpnsmiergqheeaktklrrirgvddvsqefddlvaaskesqsiehpwrnllrrkyrphltmaymipffqqltginvimfyapvlfntigfttdaslmsavvtgsvnvgativsiygvdrwgrrflfleggtqmlicqavvaacigakfgvdgtpgelpkwyaivvvtficiyvagfawswgplgwlvpseifpleirsaaqsitvsvnmiftfiiaqifltmlchlkfglflvfaffvvvmsifvyiflpetkgipieemgqvwrshwywsrfvedgeygnalemgknsnqa gtkhvSEQ ID NO 5: glucose transport protein Vicia faba from CAB07812mpaagipigagnkeypgnitpfvtitcvvaamgglifgydigisggvtsmnpflekffpavyrkknaqhsknqycqydsetltlftsslylaallssvvastitrrfgrklsmlfggllflvgalinglaqnvamlivgrillgfgigfanqsvplylsemapykyrgalnigfqlsitigilvanilnyffakikggwgwrlslggamvpaliitigslilpdtpnsmiergdrdgakaqlkrirgvedvdeefndlvaasetsmqvenpwrnllqrkyrpqltmavlipffqqftginvimfyapvlfnsigfkddaslmsavitgvvnvvatcvsiygvdkwgrralfleggvqmlicqvavaysiaakfgtsgepgdlpkwyaivvvlficiyvagfawswgplgwlvpseifpleirsaaqsvnvsvnmlftflvaqifltmlchmkfglflffaffvvvmtiyiytmlpetkgipieemdrywkshpywsrfvehddngvemakggvknv SEQ ID NO 6: Galactose-H+symporter from Q39524 Parachlorella kesslerimagggpvastttnrasqygyargglnwyifivaltagsggllfgydigvtggvtsmpeflqkffpsiydrtqqpsdskdpyctyddqklqlftssfflagmfvsffagsvvrrwgrkptmliasvlflagaglnagaqdlamlvigrvllgfgvgggnnavplylsecappkyrgglnmmfqlavtigiivaqlvnygtqtmnngwrlslglagvpailingslllpetpnslierghrrrgravlarlrrteavdtefedicaaaeestrytlrqswaalfsrqyspmlivtsliamlqqltginaimfyvpvlfssfgtarhaallntviigavnvaatfvsifsvdkfgrrglfleggiqmfigqvvtaavlgvelnkygtnlpsstaagvlvvicvyvaafawswgplgwlvpseiqtletrgagmsmavivnflfsfvigqaflsmmcamrwgvflffagwvvimtffvyfclpetkgvpvetvptmfarhwlwgrvmgekgralvaadear kagtvafkvesgsedgkpasdqSEQ ID NO 7: ATSTP2 carbohydrate transporterArabidopsis thaliana from NP_172214mavgsmnveegtkafpakltgqvflccviaavgglmfgydigisgvtsmdtflldffphvyekkhrvhennyckfddqllqlftsslylagifasfissyvsrafgrkptimlasifflvgailnlsagelgmliggrillgfgigfgnqtvplfiseiaparyrgglnvmfqflitigilaasyvnyltstlkngwryslggaavpalilligsffihetpasliergkdekgkqvirkirgiedielefneikyatevatkvkspflcelftksenrpplvcgtllqffqqftginvvmfyapvlfqtmgsgdnaslistvvtngvnaiatvisllvvdfagrrcllmegalqmtatqmtiggillahlklvgp itghavrSEQ ID NO 8: yeast hexokinase from P04806mvhlgpkkpqarkgsmadvpkelmdeihqledmftvdsetlrkvvkhfidelnkgltkkggnipmipgwvmefptgkesgnylaidlggtnlrvvlvklsgnhtfdttqskyklphdmrttkhqeelwsfiadslkdfmveqellntkdtlplgftfsypasqnkinegilqrwtkgfdipnveghdvvpllqneiskrelpieivalindtvgtliasyytdpetkmgvifgtgvngafydvvsdieklegkladdipsnspmainceygsfdnehlvlprtkydvavdeqsprpgqqafekmtsgyylgellrlyllelnekglmlkdqdlsklkqpyimdtsyparieddpfenledtddifqkdfgvkttlperklirrlceligtraarlavcgiaaicqkrgyktghiaadgsvynkypgfkeaaakglrdiygwtgdaskdpitivpaedgsgagaaviaals ekriaegkslgiiga

1. A method for culturing Botryococcus braunii microalgae, the methodcomprising: providing a culture medium that includes a fixed carbonsource in a fermentor; inoculating the fermentor with a strain ofBotryococcus braunii microalgae capable of metabolizing the fixed carbonsource; culturing the microalgae in heterotrophic conditions for atleast 72 hrs. sufficient to produce growth and/or propagation of themicroalgae, wherein the fermentor does not allow light to strike themicroalgae, and wherein the dry cell weight of the microalgae increasesby at least 2-fold as a result of the culturing.
 2. The method of claim1, wherein the fixed carbon source comprises a carbohydrate.
 3. Themethod of claim 1, wherein the fixed carbon source is selected from thegroup consisting of glucose, mannose, galactose, fructose, glycerol, anda combination thereof.
 4. The method of claim 1, wherein the culturemedium is additionally provided with a complex nitrogen source before orduring culturing.
 5. The method of claim 4, wherein the complex nitrogensource is selected from the group consisting of urea, hydrolysatecasein, and a combination thereof.
 6. The method of claim 1, wherein theinoculating is performed using an inoculum of Botryococcus brauniimicroalgae that has been cultured in the dark for at least one passageprior to the inoculation.
 7. The method of claim 6, wherein the inoculumhas been cultured in the dark for a plurality of passages prior toaddition to the fermentor.
 8. The method of claim 1, additionallycomprising, after the culturing, transferring all or a portion of themicroalgae to a further fermentor, and further culturing the microalgaefor a period of time, wherein the further fermentor does not allow lightstrike the microalgae.
 9. The method of claim 1, wherein the culturingis carried out for a period of time to generate microalgal biomass; andthe method additionally comprises: extracting hydrocarbons from themicroalgal biomass to produce extracted hydrocarbons.
 10. The method ofclaim 9, wherein the method additionally comprises: separating differentspecies of the extracted hydrocarbons.
 11. The method of claim 9,wherein the extracting is performed by hexane extraction.
 12. The methodof claim 9, wherein the extracting is performed by in vivo extraction.13. The method of claim 10, wherein the separating comprises isolatingdifferent species of hydrocarbons in a fractional distillation column.14. The method of claim 1, wherein after the culturing, the dry cellweight of the microalgae is greater than the dry cell weight of the samestrain of microalgae cultured in the presence of light, with all otherculture conditions being the same.
 15. The method of claim 14, whereinthe dry cell weight of the microalgae is at least about 2-fold greaterthan the dry cell weight of the same strain of microalgae cultured inthe presence of light, with all other culture conditions being the same.16. The method of claim 1, wherein the culture medium further comprisesat least one additional component selected from the group consisting ofdextrin, malt extract, traders yeast, corn meal, corn steep powder,whole dead yeast, casein type M, casein type B, tomato paste, molasses,soy hydrolysate, non-fat dry milk, soy flour, corn starch and maltose.17. A method for culturing Botryococcus braunii microalgae, the methodcomprising: providing a culture medium that includes a fixed carbonsource; inoculating the culture medium with a strain of Botryococcusbraunii microalgae capable of metabolizing a fixed carbon source;culturing the microalgae in essentially heterotrophic conditions for aperiod of time sufficient to increase the dry cell weight of themicroalgae by at least 2-fold.
 18. The method of claim 17, wherein theculturing is carried out for a period of time to generate microalgalbiomass; and the method additionally comprises: extracting hydrocarbonsfrom the microalgal biomass to produce extracted hydrocarbons.
 19. Themethod of claim 18, wherein the method additionally comprises:separating different species of the extracted hydrocarbons.
 20. Themethod of claim 17, wherein after the culturing, the dry cell weight ofthe microalgae is greater than the dry cell weight of the same strain ofmicroalgae cultured in the presence of light, with all other cultureconditions being the same.
 21. The method of claim 20, wherein the drycell weight of the microalgae is at least about 2-fold greater than thedry cell weight of the same strain of microalgae cultured in thepresence of light, with all other culture conditions being the same.